Down regulation of Ras protein by Metal Macrocycles. Dissertation to obtain the degree. Doctor Philosophiae (Doctor of Philosophy, PhD)

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1 Down regulation of Ras protein by Metal Macrocycles Dissertation to obtain the degree Doctor Philosophiae (Doctor of Philosophy, PhD) at the Faculty of Biology and Biotechnology Ruhr-University Bochum International Graduate School in Biosciences Ruhr-University Bochum Submitted by Ravi Shankar Bojja From Hanamkonda, India Bochum January 2008

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3 1. Berichterstatter: Prof. Dr. Klaus Gerwert 2. Berichterstatter: Prof. Dr. Hermann Lübbert 3. Berichterstatter: Prof. Dr. Gerhard Link

4 Statement by the Candidate I wish to state that the work embodied in this thesis titled "Down regulation of Ras protein by Metal Macrocycles" forms my own contributions to the research work carried out under the guidance of Prof. Dr. Klaus Gerwert at the Department of Biophysics, Ruhr Universität, Bochum. This work has not been submitted for any other degree of this or any other university. Certified by: Signature of the Guide Prof. Dr. Klaus Gerwert Signature of the Candidate Ravi Shankar Bojja i

5 Dedicated to My parents, brothers and sisters for their love and care My mentors for their invaluable advices My friends for everything ii

6 Index 1 Introduction Ras Superfamily verview Ras superfamily structure Ras superfamily GTPase biochemistry and regulation Lipid modification and membrane targeting GTPase Activating Proteins (GAPs) Role of GAP RasGAP and the arginine finger in trans The Conformational Switch and GTPase GAP hydrolysis mechanism Anticancer Strategies Farnesyltransferase Inhibitors (FTIs) RNA interference (RNAi) Reovirus Therapy Targeting Ras Effector Signaling Pathway S-nitrosation of Ras superfamily GTPases FTIR on Ras Switching mechanism Summary of previous FTIR studies on Ras and T35S Aim of the thesis Characterization of Semi-Synthetic Ras protein by trftir Modulation of the Ras-effector interactions with Zinc Cyclen Substituted metal macrocycles role on GTPases activity Emulating the RasGAP activity with metal peptide bioconjugates 20 iii

7 1.7.5 S-nitrosation of the GTPases by metal nitrosyl complexes 21 2 Materials and Methods Materials Chemicals Enzyme Bacterial strains and Proteins ther Materials Instruments Columns Synthesis of Cyclen, Cyclam derivatives and metal complexes Synthesis of Zinc Cyclen Synthesis of Copper Cyclam Synthesis of L Synthesis of L Tripod pyridyl derivatives Miscellaneous complexes Synthesis of Zinc macrocyles of L1, L2, Fab Titanium Peroxocitrate Caged and Mant nucleotides Synthesis of caged GppNHp Synthesis of Mant-GDP Synthesis of Metal NF1 bioconjugate Synthesis of monosubstituted cyclam Synthesis of NF1 peptide fragment Synthesis of NF1 peptide bioconjugate Synthesis of Metal NF1 peptide bioconjugate Analytic Methods Synthesis of Metal Nitrosyl Complexes Synthesis of K 2 [RuCl 5 N].xKCl Synthesis of trans-[rucl(n)(cyclam)]cl 2.xH 2 (L4) 35 iv

8 2.8.3 Synthesis of trans-[rucln([15]anen 4 )]Cl.xH 2 (RuA) Biochemical Methods verexpression of Ras wild type, Ras T35S and Ras Y32W in E. coli Expression System Purification of Ras wild type, Ras T35S and Ras Y32W proteins from E. coli cultures Nucleotide exchange Nucleotide detection using reverse phase HPLC Time resolved infrared spectroscopy Instrumentation Basic principles of Infrared spectroscopy Sample preparation Initiation of the reaction and data collection Measurement parameters Data analysis Ab initio calculations on triphosphate models Nucleotide dissociation with metal nitrosyl and peroxo complexes Fluorescence spectroscopy / MantGDP-exchange assay Cytotoxicity experiments Cell culture conditions Cytotoxicity Assays 51 3 Results Studies on Semisynthetic Ras ff to n conformation of the semi-synthetic Ras protein NF1 activation of the semisynthetic Ras protein Modulation of Ras protein by Zinc cyclen Interaction of Zinc cyclen with Ras protein Interaction of Zinc cyclen with ff state Ras mutants GTPase activity of Ras protein in the presence of the Zinc Cyclen 65 v

9 3.3 Interaction of Ras protein with miscellaneous compounds Interaction of 1,2,3-Hexanetriol with Ras*GTP Switching with Spermidine, Spermine, Putrescene Switching by Acetonitrile (AcCN) and Dimethylsulphoxide (DMS) GTP complex formation with metal macrocycles Complex formation between GTP and Copper Cyclam Complex formation between free GTP, Copper Cyclam, Zinc derivative (L1) and Zinc Complex of Fab Free GTP hydrolysis at 353K in the presence of the compounds (L1, L2, and Fab7) GTPase activity of Ras and RasG12V in the presence of zinc substituted macrocycles Ab initio Calculations on the triphosphate models ptimizations, ESP and energies of the models Frequencies Analysis trftir studies on non-hydrolyzable GTP analogs and GDP bound to Ras protein Photolysis difference spectrum and kinetics of Ras*GppCH 2 p Interaction of Ras*GppCH 2 p with Zinc cyclen Interaction of Copper Cyclam with Ras*GDP Metal NF1 peptide bioconjugates Synthesis of the Ruthenium NF1 peptide bioconjugate trftir measurements of GTPase activity with NF1 peptide, NF1 peptide conjugate and Ruthenium NF1 peptide bioconjugate GAP assisted GTPase activity of Ras in the presence of 1% NF1 and Ruthenium NF1 peptide bioconjugate Study of switching in the presence of Ruthenium NF1 peptide bioconjugate Nitrosation of Ras protein Photolysis studies of N and 2 release from metal complexes 102 vi

10 Interaction of N, 15 N and 2 with Ras*GDP Protein bound nucleotide degradation on UV Irradiation Intrinsic GTPase activity in the presence of nitrosation environment Role of redox environment on the GTPase activity of the Ras Protein in the presence of 1-2% NF Interaction of the effector RalGDS with T35S in the presence of nitrosation environment Anticancer activity of Ruthenium nitrosyl complex Discussion Semisynthetic Ras Protein Switching of conformations with small molecules Ab initio studies and implications of non-hydrolyzable nucleotides GTPase activities with small molecules and Metal NF1 peptide biocojugates Nitrosation induced changes and associated down regulation of Ras protein Future perspectives Summary Zusammenfassung References Appendix Mass spectrum of Zinc Cyclen Sulfate UV-Vis characterization of the nitroso and peroxo metal complexes after UV irradiation a Photolysis Difference Spectrum of Titanium Peroxo Citrate b Photolysis Difference Spectra of Ruthenium Nitroso Complex PDB structures and distances between different phosphates SDS PAGE of Semisynthetic Ras protein Amplitude Spectra of AcCN and DMS interaction with Ras protein Electrostatic Potential charges of T1, T2, & T Calculated IR spectra of T1, T2, T Mass spectrum of truncated Ruthenium NF1 peptide bioconjugate Hydrolysis difference spectra of Ras and Q61A in the presence of 2X of Ruthenium NF1 peptide bioconjugate Mass spectra of the Nitro and xo products of GDP and GppNHp 152 vii

11 8.12 HPLC analysis of the Ras protein intrinsic reaction mixture flowthrough after GTPase activity Abbreviations 154 Acknowledgements 157 Lebenslauf 158 viii

12 Abstract Downregulation of Ras protein by Metal macrocycles Ravi Bojja, Ruhr Uni-Bochum, Germany. The small guanine nucleotide binding protein Ras is involved in cellular signal transduction and cell proliferation. There it acts as a molecular switch with an active GTP-bound (on) state and an inactive GDP-bound (off) state. Conformation states of p21 ras are associated with interaction of effectors regulating the signal transduction and cell proliferation. Ras bound with GTP analog (GppNHp) has been observed with two conformation states (1 & 2) as determined by NMR studies a. From NMR studies it is revealed that state 2 is an active form of GTP bound Ras with high affinity to Raf-Kinase, while state 1 is shown to be a conformation similar to GDP bound state. Stabilization of this latter state has an importance in development of antitumor strategies by regulating the interaction of effectors with active and inactive states of nucleotide bound Ras. Here ubiquitous Ras*GTP properties and its interaction with effectors were investigated in order to down regulate the signal transduction from the following strategies A) Switching mechanism, B) Acceleration of GTP hydrolysis by metal macrocycles C) Emulating GTPase Activating Protein mimics D) Photo Dynamic Therapeutic Applications on Nucleotide dissociation.. [a] M. Spoerner, T. Graf, B. König, & H.R. Kalbitzer, BBRC, 334, (2005) ix

13 1 1 Introduction 1.1 Ras Super family verview The Ras super family of small guanosine triphosphatases (GTPases) comprise over 150 human members conserved orthologs found in Drosophila, C. elegans, S. cerevisiae, S. pombe, Dictyostelium and plants (Colicelli, J., 2004). The Ras protein is the prototype of the Ras super family of small GTPases, which can be subdivided into the Ras, Rho, Rab, Arf, Rad, Ran, Rheb, Rit and Rag families share a common biochemical mechanism and act as binary molecular switches (Vetter, I., et al., 2001). All these proteins share a high degree of sequence similarity and a common three dimensional structure, called the GTP-binding (G) domain. This domain enables them to act as molecular switches cycling between two defined conformational states: an inactive guanosine diphosphate (GDP)-bound and an active guanosine triphosphate (GTP)-bound state. Small GTPases are inefficient enzymes because their intrinsic functions, the exchange of the bound GDP to the cellular abundant GTP as well as the GTP hydrolysis (GTPase) reaction are extremely low. Although similar to the heterotrimeric G protein α subunits in biochemistry and function, Ras family proteins function as monomeric G proteins. Variations in structure (Biou, V., et al., 2004), post-translational modifications that dictate specific subcellular locations and the proteins that serve as their regulators and effectors allow these small GTPases to function as sophisticated modulators of a remarkably complex and diverse range of cellular processes. Nature invented two classes of proteins that tightly regulate the cycle between the active and inactive forms of the GTPase (Boguski, M.S., et al., 1993; Vetter, I., et al., 2001). Guanosine nucleotide exchange factors (GEFs) accelerate the exchange of bound GDP for GTP, whereas GTPase-activating proteins (GAPs) stimulate the GTP hydrolysis reaction. In its active GTP bound conformation the GTPase can interact with and regulate a spectrum of functionally diverse effector proteins, participating in a network of signaling cascades.

14 Ras superfamily structure Ras superfamily GTPases function as GDP/GTP- regulated molecular switches (Vetter, I., et al., 2001). They share a set of conserved G box GDP/GTP-binding motif elements beginning at the N-terminus: G1, GXXXXGKS/T; G2, T; G3, DXXGQ/H/T; G4, T/NKXD; and G5, C/SAK/L/T (Bourne, H.R., et al., 1991) (Figure 1.1). Together, these elements make up an ~20 kda G domain (Ras residues 5-166) that has a conserved structure and biochemistry shared by all Ras superfamily proteins, as well as Gα and other GTPases. Figure 1.1 Domain structure of the Ras superfamily (Wennerberg, K., et al., 2004) Ras superfamily GTPase biochemistry and regulation Small GTPases exhibit high-affinity binding for GDP and GTP, and possess low intrinsic GTP hydrolysis and GDP/GTP exchange activities. GDP/GTP cycling is controlled by two main classes of regulatory protein. Guanine-nucleotide-exchange factors (GEFs) promote formation of the active, GTP-bound form (Schmidt, A., et al., 2002), whereas GTPase-activating proteins (GAPs) accelerate the intrinsic GTPase activity to promote formation of the inactive GDP-bound form (Bernards, A., et al., 2004) (Figure 1.2). GTPases within a branch use shared and distinct GAPs and GEFs. GTPases in different branches exhibit structurally distinct but mechanistically similar GAPs and GEFs. The two nucleotide-bound states have similar conformations but these have pronounced differences corresponding to the switch I (Ras residues 32-38) and switch II (59-67) regions (Figure 1.3). The GTP-bound conformation possessing high affinity for effector proteins like Raf, RalGDS (Bishop, A.L., et al, 2000; Repasky, G.A., et al., 2004). It is mainly through the conformational changes in these two switches that regulatory proteins and effectors sense the nucleotide status of the small GTPases. Arf proteins contain additional N-terminal sequences, whereas Ran has additional C-terminal sequences that undergo significant

15 3 conformational changes during GDP/GTP cycling. Although the GTP-bound form is the active form for all Ras superfamily GTPases, the cycling between the GDP-bound and GTP-bound states, in which distinct functions are associated with each nucleotide-bound form, is also critical for the activities of Rab, Arf and Ran GTPases. The core effector domain (Ras residues 32-40) includes the switch I domain and is critical for direct association with effectors (Herrmann, C., 2003) and mutation at residue 35 to serine renders a partial loss function Ras mutant with less affinity for effectors. Inactive Active Figure 1.2 The Ras-cycle. Ras cycles between a GDP and GTP-bound conformation. The intrinsic reactions can be catalyzed by GTPase activating proteins (GAP) and Guanine nucleotide-exchange-factors (GEF). The Ras-switch. The figure on the left side shows H-Ras with bound GDP (PDB: 1AA9), the figure on the right shows the GTP-bound conformation (PDB: 1219, GppCH 2 p bound). The nucleotide is depicted in blue, the switch I and switch II regions in red and green, respectively.

16 4 Figure 1.3 Top view of the switch regions: Crystal structure of the Ras*GTP-conformation, from the top view of the nucleotide. Switch I is colored in golden orange, Switch II is in hot pink, Mg 2+ in blue sphere, GTP is in multi color stick structure (PDB: 1QRA) Lipid modification and membrane targeting Apart from cycling between active and inactive forms of the nucleotide bound state, the other important biochemical feature of a majority of Ras superfamily proteins is their ability to undergo post-translational modification by lipids. The majority of Ras and Rho family proteins terminate with a C-terminal CAAX (C= Cys, A = aliphatic, X = any amino acid) tetrapeptide sequence (Cox, A.D., et al., 2002a, b). This CAAX motif, when coupled together with residues immediately upstream (e.g. cysteine residues modified by the fatty acid palmitate), comprises the membrane-targeting sequences that dictate interactions with distinct membrane compartments and subcellular locations. The CAAX motif is the recognition sequence for farnesyltransferase and geranylgeranyltransferase I, which catalyze the covalent addition of a farnesyl or geranylgeranyl isoprenoid, respectively, to the cysteine residue of the tetrapeptide motif. Rab family proteins terminate in a distinct set of cysteine-containing C-terminal motifs (CC, CXC, CCX, CCXX, or CCXXX) that are similarly modified by geranylgeranyltransferase II, which also attaches geranylgeranyl groups. Some members of the Arf family are modified at their N-termini by a

17 5 myristate fatty acid. These modifications are essential for facilitating membrane association and subcellular localization critical for biological activities. Rho and Rab GTPases are regulated by a third class of proteins, guanine nucleotide dissociation inhibitors (GDIs), which mask the prenyl modification and promote cytosolic sequestration of these GTPases (Seabra, M.C., et al., 2004). Some Ras superfamily members do not appear to be modified by lipids, but still associate with membranes (e.g. Rit, RhoBTB, Miro and Sar1). thers (e.g. Ran and Rerg) are not lipid modified and are not bound to membranes (Der, C.J., 2006). 1.2 GTPase activating proteins (GAP) Role of GAP The intrinsic GTPase activity of Ras proteins is accelerated by GTPase activating proteins (GAPs), which act to attenuate GTPase signaling by accelerating the conversion of bound GTP to bound GDP. Tumor associated Ras proteins harbor single amino acid substitutions at residues Gly 12 and Gln 61 that impair the intrinsic and GAP stimulated GTPase activity, thus rendering these mutant Ras proteins persistently GTP bound and active in the absence of extracellular stimuli. For the small GNBPs like Ras, Rho, Ran, Rab and Arf, specific GAPs catalyze GTP hydrolysis, which are all different at the sequence level and employ certain unifying and some divergent features to accelerate GTP hydrolysis of the cognate GNBP (Rojas, J.M., et al., 2006) RasGAP and the arginine finger in trans RasGAPs were discovered when it was found that Ras GTP microinjected in cells was rapidly converted to Ras GDP (Trahey, M., et al., 1987). The protein responsible for this activity was identified and called p120gap. A fragment of 334 residues (GAP-334) was shown to be sufficient for catalytic activity. GAP-334 itself is a helical elongated protein with a central domain of 218 residues, which is conserved among all RasGAPs (Scheffzek, K., et al., 1996). Later on, the complex structure (Scheffzek, K., et al., 1997) of Ras and GAP-334 along with AlF 3, a classical analogue for the gamma phosphate of ATP or GTP (Mittal, R., et al., 1996), revealed that the GAP interacts predominantly with the switch regions and the P-loop of Ras (Figure 1.4). A conserved arginine residue from an exposed loop of RasGAP complements the

18 6 catalytic site of Ras and the guanidium group of this arginine contacts the β-phosphate and AlF 3. In addition, the main chain carbonyl oxygen of R789 makes a hydrogen bond to the side chain amide group of the catalytic glutamine (Q61) of Ras and stabilizes its position. Due to its positioning on a flexible loop and direction towards the active site, it has been termed as Arginine finger. Figure 1.4 Transition state analogue of Ras*RasGAP*GDP*AlF x (PDB: 1WQ1) p120gap (light brown), Arginine finger (blue), Mg 2+ (Cyan, Sphere), Ras protein (aqua green), AlF 3 - (hot pink), GDP (multicolor, stick model) (Scheffzek, K., et al., 1997). Thus, the principles of GTPase stimulation by GAP were suggested to be (i) stabilization of the switch regions (ii) stabilization of Q61 leading to correct positioning of the nucleophilic water molecule and (iii) supplying a positive charge (arginine finger) to compensate the developing negative charge at the transition state when it is associative. This structure also explains why the G12V mutation of Ras is oncogenic. Glycine 12 is within van-der-waals distance of the catalytic arginine and any mutation interferes sterically with the correct transition state formation, though such mutants still bind to the GAP (Polakis, P., et al., 1993). Glutamine 61 points towards the phosphate chain of the nucleotide and is stabilized in its orientation by a hydrogen bond with the

19 7 main chain carbonyl group of the invariant Arg789. This stabilizes the transition state and mutation to any other residue-even alanine-would disturb the transition state (Scheffzek, K., et al., 1997) of the reaction. In the aforementioned cases, the active site is complemented by a catalytic arginine from a cognate protein, often termed in trans (Ahmadian, M.R., et al., 1999). Although no sequence similarity is observed, based on the structures it was proposed that RasGAPs and RhoGAPs have a common evolutionary ancestry (Rittinger, K., et al., 1998). Interestingly, bacteria have developed strategies to modulate Rho GTPase activity of host cells through toxins as Salmonella SptP (Fu, Y., et al., 1999), Pseudomonas ExoS, Yersinia YopE (Goehring, U.M., et al., 1999) and ExoT (Krall, R., et al., 2000). The conserved sequence patterns (Litvak, Y., et al., 2003) of these small proteins (~140 residues) fold into exposed loop structures (Wurtele, M., et al., 2001; Stebbins, C.E., et al., 2001; Evdokimov, A.G., et al., 2002), termed as bulges that carry the functionally important residues of these helical proteins. The described mechanism of GAP-assisted GTP hydrolysis clearly does not apply to all G- domain proteins. Rap proteins have a Thr, and protein synthesis factors a His in place of the catalytic glutamine. RapGAP uses asparagines instead of arginine for acceleration. Understanding why oncogenic mutants of Ras cannot be switched off by GAP has invoked the concept of restoring the GTPase activity of oncogenic Ras mutants as a therapeutic approach for Ras-directed cancer therapy (Wittinghofer, A., et al., 2000; Ahmadian, M.R., 2002). Apart from assistance from GAPs in GTPase reaction, the GTPase activity of the oncogenic protein is increased up to three orders of magnitude by using a modified GTP analogue, 3,4- diaminobenzophenone-phosphoramidate-gtp (DABP-GTP), instead of GTP (Ahmadian, M.R., et al., 1999). The structures of DABP-GppNHp bound to Pro12 and Val12 mutants of Ras, revealed that the DABP moiety is accommodated close to a hydrophobic patch of Pro12 or Val12 in the P-loop. DABP-GTP provides an aromatic amino group that is critical for the mechanism of DABP-GTP cleavage, which differs substantially from the intrinsic and GAPstimulated GTP hydrolysis by Ras (Ahmadian, M.R., et al., 1999; Gail, R., et al., 2001). Catalytic drugs that target the GTPase reaction may be able to complement the insensitive GAP activities in Ras transformed cancer cells and restore the defective GTPase reaction of oncogenic Ras proteins.

20 8 1.3 The Conformational Switch and GTPase GAP hydrolysis mechanism Ras structures in both the GDP- and GTP-bound form have been described (Figure 1.2), which displays the requirements of the molecular switches. Structural differences are usually confined primarily to two segments, first observed in Ras, which are called the switch regions (Milburn, M.V., et al., 1990). These regions usually show an increased flexibility in X-ray structures and in studies using nuclear magnetic resonance (NMR) and electron paramagnetic resonance (Farrar, C. T., et al., 1997, Ito Y., et al., 1997). Furthermore, whereas the GDP-bound proteins (Figure 1.2) show a large variation in structural details, the GTP-bound forms of the G domain are remarkably similar. The trigger for the conformational changes in most of the GTPases members are likely to be universal (Vetter, I., et al., 2001). Figure 1.5 Schematic diagram of the universal switch mechanism where the switch I and II domains are bound to the γ -phosphate via the main chain NH groups of the invariant Thr and Gly residues, in what might be called a loaded spring mechanism. Release of the γ-phosphate after GTP hydrolysis allows the switch regions to relax into a different conformation (Vetter, I., et al., 2001). In the triphosphate-bound form, there are two hydrogen bonds from γ-phosphate oxygens to the main chain NH groups of the invariant Thr and Gly residues (Thr35/Gly60 in Ras) in switch I and II, respectively (Figure 1.5). The glycine is part of a conserved DXXG motif; the threonine is also involved in binding Mg 2+ via its side chain. The conformational change can be best described as a loaded spring mechanism where release of the γ-phosphate after GTP hydrolysis allows the two switch regions to relax into the GDP specific conformation (Figure 1.5). The

21 9 extent of the conformational change is different for different proteins and involves extra elements for some proteins. Figure 1.6 Associative nature of GAP assisted GTPase activity. The γ-phosphate is already cleaved from the GDP. Despite the absence of a covalent bond to the phosphate, it is retained in the binding pocket via a hydrogen bond and electrostatic interactions with the magnesium atom (Kötting, C., et al., 2006). In the GAP assisted GTPase activity, the mechanism proceeds through so called substrateassisted catalysis, in which GAP-catalyzed GTPase reaction of Ras proceeds via an identifiable intermediate state and release of the phosphate is the rate determining step (Kötting, C., et al., 2006) (Figure 1.6). The intermediate state corresponds to γ-phosphate which has not yet dissociated from Ras. From trftir studies, the vibrations of the β, γ-bridging oxygen of GTP bound to Ras had demonstrated that this bridge is already broken in the intermediate. The close juxtaposition of the GDP and Pi in the intermediate state is in harmony with the finding that a considerable amount of back reaction to GTP takes place not only in Ras*RasGAP but also in the Rap*RapGAP reaction (Chakrabarti, P.P., et al., 2004) and might be a general phenomenon of this type of reactions. Studies also ruled out the possibility that the intermediate represents a pentacovalent phosphorus structure or a phosphorylated protein and FTIR bands suggest that the protein-bound Pi is a H 2 P - 4 species, possibly bound by a proton to the GDP, as shown in the

22 10 Figure 1.6. This supports a mechanism involving protonation of an oxygen of the γ-phosphate group to form an associative transition state. 1.4 Anti-cancer Strategies Anti-cancer drug discovery is a complex, time-consuming, and risky process, and the road to success is filled with unexpected twists and turns. Extensive experimental analyses in cell culture and animal models have established strong and compelling validation of a causal role of aberrant Ras function in tumor progression and maintenance. Blocking Ras function for cancer treatment has evolved into following strategies with their outcomes: A) Farnesyltransferase Inhibitors (FTIs), B) RNAi, C) Reovirus Therapy, D) Targeting Ras effector signaling pathway Farnesyltransferase Inhibitors (FTIs) Ras proteins are synthesized initially as cytosolic proteins that undergo a rapid series of posttranslational modifications that are vital to their normal and oncogenic functions (Cox, A. D., et al., 2002a,b, Sebti, S. M., et al., 2003, Winter-Vann, A. M., et al., 2005). First, farnesyltransferase (FTase) covalently attaches a farnesyl isoprenoid lipid to the cysteine located in the C-terminal CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal amino acid) that is found in all Ras proteins, in other members of the Ras superfamily, and in many other proteins. Farnesylation is the obligate first step in this process, as inhibition prevents all subsequent processing steps. FTIs, while designed initially for the purpose of inhibiting Ras function, there are not Ras-selective inhibitors. FTIs block more than one farnesylated protein simultaneously, and the relevant combination of target proteins may not be the same in every tumor type RNA interference (RNAi) This is a process in which small interfering RNAs (sirnas) are designed to complement and anneal to the mrna for a particular protein, thereby preventing translation and creating a functional knock-out of that protein. RNAi has become widely popular in the functional evaluation of proteins across many fields of research, and may also find utility as a cancer

23 11 treatment (Duursma, A. M. et al., 2003 & Dykxhoorn, D. M., et al., 2005). Preclinically, small interfering RNA oligonucleotides are delivered directly to cells in culture or animal models. Increasingly, short hairpin RNAs (shrnas) expressed from retrovirus and lentivirus plasmids are packaged for introduction into tumor cells where they are then processed into sirnas by the RNAi machinery. As with many other gene therapy-style approaches, a limitation of the RNAi approach in targeting Ras function is associated with the difficulty in delivering unstable sirnas into the tumor cells Reovirus Therapy In recent years the power of lytic viruses to efficiently infect and kill human cells has emerged as an intriguing and promising new approach to cancer treatment (Zwiebel, J. A., 2001, Norman, K. L. et al., 2005). ncolytic viruses currently under development include adenovirus, adenoassociated virus, Sindbis virus, herpes simplex virus, Epstein-Barr virus, Newcastle disease virus, and reovirus. An advantage of using viruses is their natural ability to survive in vivo, and to enter and kill cells, thus avoiding some of the major pharmacokinetic and pharmaco-dynamic problems associated with small molecule anti-cancer agents. Moreover, the use of viruses avoids the need for potentially toxic chemical compounds to kill cells because these viruses are selectively toxic to the cells they infect. But, as with any anti-cancer drug, if viruses are not made highly selective for either delivery to or killing of tumor cells vs. normal cells, then general toxicity will limit their usefulness Targeting Ras effector signaling pathway Even in cancers in which Ras is not mutated, Ras activity is often increased as a result of other genetic lesions, in particular, overexpression and/or mutational activation of receptor tyrosine kinases that function upstream of Ras. A vast of body of evidence has accumulated implicating Ras in virtually every aspect of malignant tumorigenesis, including increased cell proliferation, acquisition of anchorage independence, survival, motility and invasion, and metastasis (Hanahan, D., et al., 2000, Campbell, P. M., et al., 2004). Collectively these observations have made Ras an attractive target for anti-cancer drug development efforts (Cox, A. D., et al., 2002a, b; Downward, J., 2003). Naturally occurring mutations of Ras are typically found at positions 12,

24 12 13 or 61, rendering Ras proteins resistant to inactivation by GAPs, while, mutations in the effectors have implications in the development of the cancer (Figure 1.7). Ras* BAY (Sorafenib) Raf* PI3K* RalGEF Tiaml* PLCs PD PD ARRY MEK AKT Ral* Rac* DAG ERK* Bad RalBPI PAK PKC Figure 1.7 Ras effector pathways involved in oncogenesis. The best characterized effectors are the Raf serine/threonine kinases, which activate the MEK>ERK MAPK cascade. The next best characterized effectors are the p110 catalytic subunits of PI3K. *Mutated, overexpressed, or persistently activated in human cancers (Fiordalisi, J.J., et al., 2006). Alternate approach of blocking oncogenic Ras function involves inhibition of Ras mediated signaling like the effectors (Figure 1.7). The initial discovery of the Raf serine/threonine kinases (c-raf-1, A-Raf, and B-Raf) as key effectors of Ras signaling and transformation identified as a promising direction for the development of inhibitors of the Raf-MEK-ERK protein kinase cascade. Studies in rodent fibroblasts established that this signaling pathway is sufficient and necessary for Ras-mediated transformation (Repasky, G. A., et al., 2004). The recent identification of mutationally activated B-Raf in a non-overlapping pattern with that of mutationally activated Ras in melanoma and in colon and other human carcinomas provided further validation of the key role of this effector pathway in Ras-mediated oncogenesis (Garnett, M. J. et al., 2004). Thus, while Ras also utilizes a multitude of other effectors, the Raf>MEK>ERK pathway has attracted the greatest interest and has seen the most significant progress to date in terms of anti-cancer drug discovery (Sebolt-Leopold, J. S. et al., 2004). Phase I-II clinical trials have been reported for kinase inhibitors of both Raf and MEK (Figure 1.7).

25 13 Currently, clinical trials of only one Raf inhibitor have been reported. The bi-aryl urea BAY was identified in a screen for inhibitors of the serine/threonine kinase p38 MAPK, and was developed originally as an inhibitor of Raf-1 (Lyons, J. F., et al., 2001). It exhibited anti-tumor activity against various human tumor cell lines, including colon, lung, breast, ovarian and pancreatic carcinomas, and melanomas, in cell culture and in mouse models (Wilhelm, S. M., et al., 2004). Although tumor inhibition was associated with inhibition of ERK activity, BAY also has significant activity against other protein kinases. In particular, BAY potently inhibits several receptor tyrosine kinases involved in tumor angiogenesis, including the vascular endothelial growth factor receptors (VEGFR) 2 and 3, the platelet-derived growth factor receptor (PDGFR), Flt-3, and c-kit. Thus, the anti-tumor activity of BAY may be due to inhibition of Raf, of angiogenesis-related kinases, or of yet other non-raf kinase targets. The anti-tumor activity of BAY in RCC (Renal Cell Carcinoma) may be due to inhibition of targets that regulate angiogenesis, and not due to inhibition of Raf. Based on these promising results, phase III trials in patients with advanced RCC are ongoing. The development and application of more potent and selective Raf inhibitors will be needed before it can be determined whether Raf is a good therapeutic target for B-Raf- and Ras-mutation positive cancers. In addition to Raf, there is evidence for the role of other effectors in promoting Ras-mediated oncogenesis (Repasky, G. A., et al., 2004) (Figure 1.7). PI3K activation of AKT has been shown to be important for oncogenic Ras function, for example, in protecting against anoikis (apoptosis in response to deprivation of matrix attachment) (Khwaja, A., et al., 1997). The recent identification of mutationally activated p110 alpha catalytic subunits of PI3K in human tumors, as well as the long-appreciated and very common loss of the tumor suppressor PTEN (a negative regulator of PI3K) in many cancers (Steelman, L. S., et al., 2004), support the important contribution of aberrant PI3K activation in cancer development and growth (Samuels, Y., et al., 2004). Hence, inhibitors of the AKT serine/threonine kinase may also be useful for blocking this important survival pathway of Ras. Members of another effector family, RalGEFs, which are guanine nucleotide exchange factors that activate the Ral small GTPases, have recently been implicated in Ras-mediated oncogenesis (Feig, L. A., 2003). RalGEF, rather than Raf or PI3K, activation was found to be sufficient to mimic Ras transformation of human cells (Hamad, N. M., et al., 2002). Ral GTPase function was

26 14 found to contribute to tumor cell anchorage-independent growth and cell survival (Chien, Y., et al., 2003). Persistent activation of RalA was found in pancreatic cancers and was important for the anchorage-independent and tumorigenic growth of these and other human cancer cells (Lim, K. H., et al., 2005). Despite intensive investigation, no strategies have yet been devised that can selectively block oncogenic Ras function in cells or animals. 1.5 S-nitrosation of Ras superfamily GTPases While the basic mechanisms by which GTPase regulatory proteins regulate GTPase substrates have been revealed through numerous studies, detailed studies into the mechanism(s) of free radical-mediated GTPase regulation have been tackled recently. Reactive Nitric oxide Species (RNS) and Reactive xygen Species (RS) influence the protein modifications such as S- nitrosation through free radical-mediated regulation, which involve nucleotide dissociation from GTPases (i.e. Ras and RhoA). Nitric xide (N) can promote formation of a cysteine thiol radical, which alters Ras activity through a radical propagation mechanism leading to guanine nucleotide oxidation and release of the modified GDP or GTP from the Ras protein (Figure 1.8). This, in turn, can lead to activation or inactivation of Ras depending on the concentration of N and oxidizing potential of the medium. S-nitrosation of Ras does not alter its structure or biochemical activity (Williams, J.G., et al, 2003), However, N can also regulate the activity of proteins by mechanisms that are independent of S-nitrosation. Recent studies on the Ras GTPase indicate that it is cysteine thiol radical formation rather that S-nitrosation, that alters Ras activity (Heo, J., et al., 2006 & 2005a). The spatial orientation of the Phe28 and Cys118 side chains (Ras numbering) within the NKCD motif of Rap1A and Rab3A are similar to as observed in the Ras protein NKCD motif (Heo, J., et al., 2005). Similarly, all Rho subfamily GTPases are regulated by redox species, that contain a cysteine, Cys18 (Rac1 numbering), located at the end of the P-loop is designated the GXXXXGK(S/T)C motif. While the members of the Ras subfamily of GTPases (i.e., H, K, N Ras, Rab3A and Rap1A) possess a NKCD motif which forms interactions with the guanine nucleotide base and contains a redox-active cysteine, Cys118 (Ras numbering) (Heo, J., et al., 2005, 2005a, 2004, Lander, H.M., et al, 1996 ). For the RhoA protein, which is the member of the Rho subfamily also stimulates guanine nucleotide dissociation with a similar mechanism like Ras but it is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine

27 15 nucleotide binding and is permanently inactivated by RNS / RN species (Heo, J., et al., 2005b). Challenges remain to better characterize conditions by which Ras is activated or inactivated by redox agents in various cellular conditions. Current interest is to understand how Ras GEFs and GAPs cooperate in the presence of redox agents. Inhibition of GTPases properties like interaction with effectors under low reducing potentials or S-nitrosation of the C-terminus of the Ras protein opens an option of using N as an anticancer agent. R R N N N N N H Cys 118 H Phe 28 S H H R HN Asp 119 R R N N N 2 Nlm-DP H N HN N Cys 118 H H H S R - HN R Phe 28 Asp 119 R RNS / RN R RNS / RN R Phe 28 N N N N H N H H Asp 119 Cys 118 S R HN Figure 1.8 Proposed radical based mechanism of redox mediated Ras guanine nucleotide dissociation. Reaction of N in the presence of 2 or N 2. with the Ras Cys 118 thiol, produces a Ras radical intermediate, proposed to be a Ras thiyl radical (Ras Cys 118. ). Ras Cys 118. then electronically interacts with Ras-bound GDP, possibly via the Phe 28 side-chain, to initiate radical based conversion of Ras-bound GDP into a Ras-bound GDP neutral radical (G. DP). Ras-bound G. DP can further react with an additional. N 2 to produce GDP N 2 (5-nitro-GDP). As formation of GDP +. (electron deficient species) and sequential transformation to a G. DP will disrupt key hydrogen bond interactions between Ras residues in the NKCD/SAK motif and the guanine nucleotide base (Raines, K.W., et al., 2007). R

28 FTIR on Ras Switching mechanism Time resolved Fourier transform infrared (trftir) difference spectroscopy has proven to be a valuable tool in elucidation of reaction mechanisms at atomic resolution (Gerwert, K., et al., 1990; Allin, C., et al., 2001a, b; Kötting, C., et al., 2005). The processes which can be monitored by trftir range from conformation changes, protonation states and also bond formation and bond breakage. Labeling a specific group gives the precise information on the specific group involvement in fundamental biological processes. For the small GTPases like Ras protein the reaction mechanism of GTP hydrolysis has been studied extensively (Allin, C., et al., 2001a, b; Kötting, C., et al., 2006) and caging the GTP allows the control of the reaction until measurements are initiated by laser photolysis. In the rapid scan mode it allows the measurements of fast reactions like GAP activation of the Ras GTP hydrolysis. n the other hand at low temperatures, the reaction without GAP gives the information about the surface changes of the Ras protein during the course to attain its active conformation after decaging the phpgtp known as Switching mechanism (Kötting, C., et al., 2007). The differences of the absorption of Ras on and Ras off are calculated from the difference of the WT photolysis spectrum (Ras on GTP _ Rascaged GTP) and the Ras T35S photolysis spectrum (Ras off GTP _ Rascaged GTP). This double difference spectrum shows the conformation changes between n and ff states. (Ras on GTP _ Rascaged GTP) _ (Ras off GTP _ Rascaged GTP) = (Ras on GTP _ Ras ff GTP) Summary of previous FTIR studies on Ras and T35S In the Ras switching mechanism with phpgtp, the photolysis of caged GTP is 500ps without any intermediate states, the attained state after the photolysis is known as ff state. In the ff state, switch regions are open and threonine 35 has not coordinated to γ-phosphate and Mg 2+. The transition of wild type from ff state to n state is followed with a kinetic rate of 5-7 sec -1 and increase in the γ-phosphate vibration intensity at 1144 cm -1. The changes observed with Ras GTP n state and state before decaging of the nucleotide is shown by Photolysis difference

29 17 Figure 1.9 Switching differences of T35S: Section of the photolysis spectrum of Ras WT (black), Ras T35S (red) and Ras T35S + RafRBD (blue) showing the absorptions of the β- and γ- phosphate of the equilibrated Ras*GTP state as positive bands. NPE-caged GTP was used (Kötting, C., et al., 2007). spectrum (Figure 1.9). The transition of the T35S, ff state mutant to n state is attained after interaction with effector protein RafRBD with a rate constant of 5 sec -1. The amplitude difference spectrum between n and ff state gives the key information about the surface changes involved during the switching process (Figure 1.10). The changes observed during the transition of the ff state to n state as described in the Figure 1.10 shows some key information about the nature of GTP interaction with switch regions. The positive peaks from the spectrum represent the generation or appearance of the n state, while the negative peaks represent the dissapearence of the ff state. Key features include the increase in the intensities of the α-, β-, and γ-phosphate vibrations, while closing of the switch region of the protein is shown in the amide regions. The isotopic labeling of threonines identified the closing of switch I region by the Thr-35 backbone amide I vibration at 1689 cm -1 (Kötting, C., et al., 2007).

30 18 Figure 1.10 Double-difference spectrum between Ras on GTP and Ras off GTP, Positive peaks are due to the Ras on GTP state, negative to the Ras off GTP state. The wavenumbers for the most intense bands of both states are given (Kötting, C., et al., 2007). 1.7 Aim of the thesis Focus of the present work is followed in the respective areas of 1) Characterization of Semi-Synthetic Ras protein by trftir. 2) Modulation of the Ras-effector interactions with Zinc Cyclen. 3) Substituted metal macrocycles role on GTPases activity. 4) Emulating the RasGAP activity with metal peptide bioconjugates. 5) S-nitrosation of the GTPases by metal nitrosyl complexes Characterization of Semi-Synthetic Ras protein by trftir The contributions from amino acid sidechains are of vital importance in the progress of biological turnovers. In small GTPases, GTP hydrolysis is closely regulated by the presence of critical residues around the GTP namely P-loop, Switch I and Switch II. Mutations at the residues G12V, G13V, Q61A result in the decrease of the intrinsic GTPase activity, while such mutations are not downregulated by GAP proteins. Mutations at Switch I (T35S, Y32W) and

31 19 Switch II (A59G, G60A) adapt an ff conformation with partial loss of function. In the present study, characterization of Semisynthetic Ras protein was the main aim to establish a control system in studying the mechanism of the GTPase activity. Site-specific labeling of the essential residues like K16, T35 and Q61 (Figure 1.11) were considered for elucidation of the GTPase mechanism. Semisynthetic proteins were made by application of protein semisynthetic technique (Dr. Christian Becker, MPI, Dortmund). Q61 K16 T35 Figure 1.11 Representation of Ras protein (PDB: 1QRA), residues under proposed study are K16 (Blue), Q61 (Pink), T35 (Turquoise), GTP is in stick model, while Mg 2+ is represented in a hot pink sphere Modulation of the Ras-effector interactions with Zinc Cyclen When proteins require different conformations for their biological function, all these functional states have to coexist simultaneously in solution. However, the corresponding Gibbs free energy differences are usually rather high and thus the conformation with lowest energy predominates in solution whereas the populations of the states with higher energy (excited states) are very small.

32 20 A stabilization of these excited states can be used as a novel principle to influence the activity of proteins by small molecules. Recently 31 P- NMR studies on the Ras protein with GppNHp has shown that a small molecule like zinc cyclen can selectively stabilize the weak-binding state (Spoerner, M., et al, 2005) (Figure 1.12). The same process was monitored by trftir on Ras GTP, which is natural nucleotide with biological importance. Figure 1.12 The Ras-effector interaction in activated Ras and its modulation by small molecules by inhibition of the Ras-effector interaction by stabilization of the weak-binding state 1 (Spoerner, M., et al., 2005) Substituted metal macrocycles role on GTPases activity Substituted metal macrocycles have a tendency to accelerate the phosphate hydrolysis, here an attempt is made to study whether same phenomena is applicable in acceleration of the GTP hydrolysis bound to GTPase protein Emulating the RasGAP activity with metal peptide bioconjugates The search for peptide mimics which can catalyze the hydrolysis of the protein bound nucleotide is one of the most fascinating aspects of drug research and carries importance in the treatment of diseases. In the Ras superfamilies, GTPase activating proteins commonly known as GAPs, assist in the acceleration of the protein bound GTP hydrolysis in a multifold range (Ahmadian, M.R., et al., 1997, 1999). ne source of inspiration for drug discovery is the resolved crystal structures of these GAPs, which reveals the spatial arrangement of functional groups in the active state of

33 21 enzymes. Typical examples being the NF1 (Ahmadian, M.R., et al., 1997) and RapGAP (Daumke,., et al., 2004), each harbor unique critical amino acid residue orientated directly in the vicinity of the phosphate moiety. For NF1, arginine 789 forms a finger orientation of the side chain guanidinium results in the stabilizing the transition state of the GTPase reaction, while assumption of Rap1GAP employs a catalytic asparagine 290 directly stabilizing the transition state of GTP hydrolysis. Dual nature of the GAP activity recently reported with GAP1 (Yarwood, S., et al., 2006, Kupzig, S., et al., 2006) adds a new dimension in the understanding of the signaling pathway, where one activating protein has dual capacity to downregulate Ras as well as Rap1 protein. In cancer where GTPase proteins have mutation lacking GTP hydrolytic activity and are insensitive to GAP stimulation. The design and synthesis of the compounds with the catalytic residues involved in the acceleration of GTPase activity is a challenging goal. Metal peptide bioconjugates (Kirin, S.I., et al., 2005) consist of the peptide fragments coupled via a linker to macrocycle with a coordinated metal center. Depending on the choice of the metal these conjugates have a tendency to form affinities for carboxylic acids (Fredericks, J.R., et al., 1996) or cysteine residues (Franco, R., et al., 2001) of the proteins. Hypothesized and synthesized metal conjugates are themed to orient in a similar fashion as native GAP catalytic residues. Most of the enzyme models for phosphoryl transfer involve a typical motif arginine. For example, in the X-ray crystal structures of staphylococcal nuclease (Cotton, F.A., et al., 1979) and also in NF1 (Ahmadian, M.R., et al., 1997) protein, arginine is involved in the activation of the anionic phosphate moiety towards nucleophilic attack by water with ion-pair stabilization by the cationic guanidinium moiety. Metal macrocycle (L), which is coupled by a benzyl linker to the NF1 catalytic sequence of Gly- Gly-Gly-Thr-Leu-Phe-Arg 789 -Ala-Thr-Thr-Leu-Ser-Arg is shown in the Figure Here catalytic sequence of the NF1 which contains Arg 789 residue in the resulting metal peptide bioconjugate is studied by trftir S-nitrosation of the GTPases by metal nitrosyl complexes Under redox conditions, Ras protein has a tendency to dissociate its bound nucleotide. Here Ruthenium nitroso complex in combination with titanium peroxo citrate were studied to emulate the conditions before S-nitrosation of the Cys-118 residue on the Ras protein in vitro.

34 22 Arginine Extension Metal Complex NH X M N Gly Gly Gly Thr Leu Ph e Arg 789 Al a Thr Thr L eu NH X H N Ser Arg Figure 1.13 Representative superimposed metal peptide bioconjugate of NF1 catalytic sequence containing Arginine 789, where M is Ruthenium and X is HH or Cl - (golden orange). Loop is the NF1 sequence of interest with Arg 789 shown in blue backbone.

35 23 Application of this study could be formulated into development of photodynamic therapeutics (PDT) (Figure 1.14) to treat cancer. Apart from anticancer activity, the study could be utilized for the first time to characterize the Ras protein interactions with effectors and GAPs under redox conditions produced by nitric oxide initiated free radical propagation. Antibody/Aptamer Drug Drug Drug Receptor Normal cell Cancer cell Light Delivery Normal cell Unaffected N. 2. N. 2. Radical Formation / Apoptosis Unaffected Figure 1.14 Representation of mode of Photodynamic therapeutic (PDT) action: the schematic figure shows the delivery of the drug under the light irradiation results in selective cell death on the specific light irradiated portion.

36 24 2 Materials and Methods 2.1 Materials Chemicals Acetonitrile Acrylamide Ammonium peroxo disulfate Ammonium sulfate Ampicillin Baysilone Paste Bromophenol blue BSA Chloroform Coomassie Brilliant Blue G-250 Copper Chloride Cyclam Cyclen D 2 Diethylether N,N -Dimethylformamide DMS 1,4-Dithiothreitol EDTA Ethanol Disodium Guanosine-5 -diphosphate Trisodium salt of Guanosine-5 - triphosphate Hefe extract Hepes Hydrochloric acid Merck (Darmstadt) Fluka (Buchs, CH) Merck (Darmstadt) Merck (Darmstadt) Sigma (Dreisenhofen) Bayer (Leverkusen) Merck (Darmstadt) Serva (Heidelberg) Baker (Deventer, NL) Merck (Darmstadt) Merck (Darmstadt) Sigma (Dreisenhofen) Sigma (Dreisenhofen) Deutero (Kastellaun) Merck (Darmstadt) Baker (Deventer, NL) Baker (Deventer, NL) Biomol (Hamburg) Merck (Darmstadt) Baker (Deventer, NL) Fluka (Buchs, CH) Fluka (Buchs, CH) Gibco BRL (Paisley, GB) Sigma (Deisenhofen) Merck (Darmstadt)

37 25 Isopropyl-β-D-thiogalactopyranoside Kanamycine Low-Range Marker Lupeol Magnesium chloride Magnesium sulfate Manganese(IV)-oxide (activated) 2-Mercaptoethanol MES Methanol Baker Potassium dihydrogenphosphate Potassium hydroxide Protected amino acids, peptide synthesis resin & reagents Ruthenium(III) Chloride,xH 2 Sodium chloride Sodium hydroxide Sodium dodecylsulfate SDS Tetrabutylammoniumbromide Tetramethylendiamine Triethylamine Tris Zinc Chloride Zinc Sulfate Biomol (Hamburg) Serva (Heidelberg) Bio-Rad (Hamburg), Roth Sigma (Germany) Deventer, NL Deventer, NL Merck (Darmstadt) Merck (Darmstadt) Biomol, Hamburg Deventer, NL Baker (Deventer, NL) Baker (Deventer, NL) Merck Novabiochem (Germany) Strem Chemicals (MA, USA) Baker (Deventer, NL) Fluka (Buchs, CH) Roth (Karlsruhe) Merck (Darmstadt) Riedel-de Haën (Seelze) Fluka (Buchs, CH) Biomol (Hamburg) Merck (Darmstadt) Riedel-de Haën (Seelze) Enzyme Alkaline Phosphatase (AP) (EC ) Boehringer (Mannheim)

38 Bacterial strains and Proteins E. coli CK600K Plasmid of ptac c-h-ras and its mutants T35S, Y32W Prof. Dr. A. Wittinghofer (MPI of Molecular Physiology, Dortmund) Ral-GDS, Raf-RBD proteins, Ras*MantGDP Prof. Dr. Christian Herrmann (Ruhr Univeristat, Bochum) NF1 protein Dr. Christian Becker (MPI of Molecular Physiology, Dortmund) Ras G12V protein Prof. Dr. A. Wittinghofer (MPI of Molecular Physiology, Dortmund) ther Materials BaF 2 -Windows CaF 2 -Windows DEAE Sepharose CL-6 Dowex 50-X8 (H+)-Form Gelfiltration Desalting Korth (Kiel) Korth (Kiel) Pharmacia (Freiburg) Fluka (Buchs, CH) NAP-5 Pharmacia (Freiburg) Instruments Beckmann System Gold BioPhotometer Protein concentrators (10 kda cutoff) FT-IR-Spektrometer IFS 66 v/s DS-Hypersil C18 RP, 5 µm (250 mm x 5 mm) AkTA Florimeter Beckmann (München) Eppendorf Amicon Corporation (Ireland) Bruker (Karlsruhe) German Engineering GE Perkin Elmer

39 Columns C-18 Ultrasphere DEAE-Sepharose Fast Flow Superdex 75 und 200 Beckmann (Unterschleißheim) Amersham-Pharmacia (Freiburg) Amersham-Pharmacia (Freiburg) 2.2 Synthesis of Cyclen, Cyclam derivatives and metal complexes Synthesis of Zinc Cyclen (A) Method 1 Cyclen-Zn (II) complex was prepared by stirring at room temperature for 2 h the mixture obtained by adding slowly a solution of ZnCl 2 (52 mg, 0.4 mmol) in CH 3 H (10 ml) to a solution of cyclen (69 mg, 0.4 mmol) dissolved in CH 3 H (10 ml), then evaporation of the solvent in vacuo to give Zn(II):Cyclen (Min Su Han, et al., 2003). The crystals were redissolved in 1ml of 100 mm HEPES (ph 7.5). Method 2 Cyclen (0.09 g, 0.54 mmol) and zinc (II) sulfate (0.062 g, 0.5 mmol) were separately dissolved in MeH (15 ml) and the solutions combined. The resulting suspension was refluxed for 30 min, the mixture was evaporated to dryness and the amorphous solid was crystallized from methanol as a colorless crystalline solid (Kruppa, M., et al., 2006). The crystals were redissolved in 1 ml of 100 mm HEPES (ph 7.5) (Mass spectrum is shown in the Appendix 8.1). NH HN NH HN Zn 2+ Cu 2+ NH HN NH HN A B

40 Synthesis of Copper Cyclam (B) Cyclam (400 mg, 2 mmol) was dissolved in methanol (50 ml), and CuCl 2 (340 mg, 2 mmol) was added. The reaction mixture was heated to reflux, stirred for 2 h, and then filtered to give a clear, purple solution. The solvent was removed in vacuo, and the purple solid was recrystallized by slow diffusion of methanol into ether to give purple needle crystals of Copper cyclam (Hunter, T.M., et al., 2005) Synthesis of N, N, N, N-1,4,8,11-Tetra(hydroxypropyl)-1,4,8,11- tetraazacyclotetradecane (L1) L1 was prepared using Cyclam (4.0 g, 20 mmol) dissolved in methanol (100 ml). This solution was brought to reflux and methyl acrylate (7.56 g, 96 mmol) added dropwise. This was refluxed for a further 2 h. The methanol and excess acrylate were evaporated and the resulting oil taken up in dry THF (100 ml). Lithium aluminium hydride (6.21 g, 164 mmol) was added and the suspension refluxed with stirring for 2 h. The reaction mixture was quenched with water (6.2 ml); 15% sodium hydroxide (6.2 ml) and excess water (18.6 ml), filtered and evaporated to dryness under reduced pressure. The resulting white solid was recrystallised using ethanol to yield a white crystalline solid which was washed with diethyl ether and air-dried (Channa, A., et al., 2005). The final product consists of series of ligands from mono to tetra substitution. The quantification of the ligands synthesized was not possible to ascertain the ratio between them from mass spectra intensities, as ratio of abundance from mass spectra has no relation to composition of the ligands. Due to lack of the chromophores, these ligands are colorless and HPLC profile was difficult to assess the purity of the ligands Synthesis of N, N, N, N-1,4,8,11-Tetra(carbamoylethyl)-1,4,8,11- tetraazacyclotetradecane (L2) Similar to L1, L2 was prepared using Cyclam (0.15 g) dissolved in absolute ethanol (10 ml). This solution was brought to reflux and acrylamide (0.10 g) added dropwise (8 ml). This was further refluxed for 4 h. The ethanol was evaporated and the resulting oil taken up in dry THF (100 ml). The resulting white solid was recrystallised using ethanol to yield a white crystalline solid which was washed with diethyl ether and air-dried (Channa, A., et al., 2005).

41 29 H H H 2 N NH 2 N N N N N N N N H L1 H H 2 N NH 2 L2 The final product consists of series of ligands from mono to tetra substitution. The quantification of the ligands synthesized was not possible to ascertain the ratio between them from mass spectra intensities, as ratio of abundance from mass spectra has no relation to composition of the ligands. Due to lack of the chromophores, these ligands are colorless and HPLC profile was difficult to assess the purity of the ligands. 2.3 Tripod pyridyl derivatives Following ligands were synthesized by Fabrizio Mancin, University of Milan, Italy and named as Fab in this study: N N N Fab 1 N N N H Fab 2 N NH 2 N N N N N N N N H H NH NH 2 NH 2 NH2 2 Fab 3 Fab 4 Fab 5 N N N NH HN HN Fab 6 HN H 2 N N N H N NH C Fab 7 HN N H NH 2

42 Miscellaneous complexes Synthesis of Zinc macrocyles of L1, L2, Fab Zinc complexes of L1, L2 and Fab7 were synthesized similarly as described above (Hunter, T.M., et al., 2005, Kruppa, M., et al., 2006) with slight excess of the ligand compared to metal salt. The resulting complexes were water soluble. For the mixture of ligands, the ratio of 2:1 was maintained between ligand and metal salt to ensure maximum metal coordination to ligands. Experiments were carried out without further characterization as these metal complexes are known to bind metal tightly (Hunter, T.M., et al., 2005) Titanium Peroxocitrate Titanium peroxocitrate is provided by Markus Rohe, Lehrstuhl für Anorganische Chemie I, Ruhr-Univeristat, Bochum, which was synthesized as described (Dakanali, M., 2003). UV characterization (Appendix 8.2) and IR characterization (Appendix 8.3.a). 2.5 Caged and Mant nucleotides Caged GTP s (NPE-caged GTP and php-caged GTP) caged GDP (NPE-caged GDP) were provided by Dr. Yan Suveyzdis. Caged GppCH 2 p was commercially available (Jena Biosciences, Jena, Germany) Synthesis of the 1-(2-nitrophenyl)ethyl p3 ester of 5 -guanylyl imidodiphosphate (caged GMP-PNP) Following the general protocol (Walker, J., et al., 1988) was applied for the synthesis of caged AMP-PNP. 6 mg of the sodium salt of GMP-PNP (Fluka) in 1.0 ml of water was adjusted to ph 4.0 with dilute HC1 and stirred for 19 hr with 5 ml of freshly prepared l-(2- nitrophenyl)diazoethane in CHC1 3 extract which was filtered from the reaction of Mn 2 (Merck) with hydrazone derivative of 2-nitroacetophenone. Caged nucleotide (GppNHp) was obtained in 20% yield through purification by DEAE Sepharose (Pharmacia) pre-equilibrated with 10 mm triethylamine bicarbonate (TEAB) buffer at ph 7.5, and eluted with a linear gradient of TEAB from l0 mm to 700 mm at a rate of 60 ml. h - 1. The purified caged nucleotide was

43 31 analyzed by HPLC with retention time of 11 min on a constant gradient with 25% acetonitrile, further it was also analyzed by ESI and stored at C Synthesis of Mant-GDP Synthesis of mant-gdp was carried similar to the published (Lenzen, C., et al., 1995). 50 mg of GDP was dissolved in 2 ml of H 2 in a 15 ml Corex tube with a magnetic stirring micro-bar. ph was adjusted to 9.6 with 1 N NaH and solution was incubated at 38 C on a water bath with stirring. Synthesis reaction was started by adding 55 mg of finely powdered methylisatoic anhydride (Sigma) while maintaining ph at 9.6, and temperature at 38 C. After 1 hour, extra 53 mg of methylisatoic anhydride was added with continued incubation and stirring for one more hour. The reaction mixture was centrifuged at 5000 rpm for 10 minutes and solution was separated. The supernatant was extracted twice with chloroform from the aqueous reaction solution. The aqueous reaction mixture was purified after adjusting the ph to 7.5 with 1 N acetic acid on a DEAE Sepharose (Pharmacia) pre-equilibrated column with 10 mm triethylamine bicarbonate (TEAB) buffer at ph 7.5. The purification was done by eluting with a linear gradient of TEAB from l0 mm to 500 mm at a rate of 60 ml.h - 1. The nucleotide was analyzed by HPLC, ESI and quantified by measuring its.d. at 252 nm, 1 µm of each nucleotide should have an.d. of and further stored at -70 C. Yield was around 20% of the starting material GDP with 90 % purity. 2.6 Synthesis of Metal NF1 bioconjugate Synthesis of monosubstituted cyclam, (1,4,8,11-tetraaza- cyclotetra decane -1-(r-1,4-methylene-benzoic-acid) Monosubstitution of cyclam was carried out following the procedure as described earlier (Motekaitis, R.J, et al., 1996). 1,4,8,11-Tetraazacyclotetradecane hydrochloride (0.50 g, 2.52 mmol) was dissolved in 3.5 ml of H 2, and the ph was raised to around 8 with the small addition of LiH (Sigma). To this solution was added 15 ml of EtH, followed by p- bromomethyl benzoic acid (100.3 mg, 0.49 mmol, Sigma), and the reaction mixture was stirred at 60 C for 3 h. The EtH was removed by rotary evaporation and the aqueous phase exhaustively extracted with CHCl 3 (7 X 10 ml). The resulting chloroform extract was pooled and

44 32 allowed to evaporate overnight in the hood. Pale yellow crystalline solid is formed with yield around 70% with respect to alkylating agent and labeled as L3. Mass analysis performed by Bruker Daltonics (ESI+). The purity of the monoderivative is around 90%. NH HN 60 0 C, 3 hr NH N - NH HN p-bromomethyl benzoic acid NH HN 70% L Synthesis of NF1 peptide fragment Gly-Gly-Gly-Thr-Leu-Phe-Arg Ala-Thr-Thr-Leu-Ser-Arg Synthesis of NF1 peptide fragment constituting critical residue Arginine 789 is carried with microwave assisted solid phase synthesis by CEM Microwave peptide synthesizer Discovery system. All the amino acids were Fmoc protected from Merck Novabiochem, while Arg was protected by Pbf. The advantage of using microwave energy is the faster reactions and increased yields, which is due to extended peptide surfaces resulting from brownian motion under influence of microwave energy (Scheme 1). The NF1 peptide fragment was prepared by Fmoc solid phase synthesis, using a Rink Amide AM resin LL (Novabiochem) with an acid labile linker (Scheme 2, Kirin, S.I., et al., 2005). The peptide synthesis cycle was composed of Fmoc deprotection by piperidine and TBTU coupling (Novabiochem). After the last amino acid coupling, the resulting resin without deprotection was stored in DMF at 4 0 C. A small part of the resin was cleaved to identify the synthesized peptides by ESI.

45 33 Scheme 1 Use of microwave energy for solid phase peptide synthesis, under microwave energy, peptide surfaces are extended due to Brownian motion. Scheme 2 Solid phase synthesis of Metal NF1 peptide bioconjugate (Kirin, S.I, et al., 2005) Linker Fmoc Rink resin a,b Solid support Fmoc Arg Rink resin Pbf repeat a) and b) 12 times Fmoc-Gly-Gly-Gly-Thr-Leu-Phe-Arg-Ala-Thr-Thr-Leu-Ser-Arg Pbf Pbf Rink resin a) and c) C 10 N 4 H 23 -CH 2 C 6 H 4 C-Gly-Gly-Gly-Thr-Leu-Phe-Arg-Ala-Thr-Thr-Leu-Ser-Arg Pbf Pbf d) Rink resin C 10 N 4 H 23 -CH 2 C 6 H 4 C-Gly-Gly-Gly-Thr-Leu-Phe-Arg-Ala-Thr-Thr-Leu-Ser-Arg a) Fmoc-deprotection: 20% piperidine/dmf (2 and 10 min) b) coupling: monomer / TBTU / HBt / DIPEA (1 min) / DMF (20 min) c) coupling : ligand L3 /TBTU /HBt/ DIPEA (1 min) / DMF (20 min) d) final cleavage and side chain deprotection: TFA:TIS:H 2 (95:2.5:2.5). All reagents for peptide synthesis are from Merck, Novabiochem.

46 Synthesis of NF1 peptide bioconjugate Following the Scheme 2, a) and c) excess concentration of L3 (5 equivalents) was used to continue the coupling between NF1 peptide fragment bound to resin. After allowing sufficient coupling reaction, the excess of reagents were flushed out of the column and washed with DMF continuously to remove unbound or uncoupled ligand L3. Final cleavage from the resin and side chain deprotection was performed with TFA:TIS:H 2 (95:2.5:2.5) followed by ether precipitation of the bioconjugate to separate the peptide portion from chemical reagents and protective groups. The precipitated peptide was centrifuged, washed with ether and redissolved in 1:1 water/acetonitrile for HPLC analysis Synthesis of Metal NF1 peptide bioconjugate The NF1 peptide bioconjugate metal complex was prepared by mixing 20 ml of MeH, 50 mg of RuCl 3.xH 2 with purified NF1 peptide bioconjugate mixture fraction (2 mg), and refluxed at 80 0 C temperature for 2 hr. After evaporation of the alcohol, the resulting metal bioconjugate mixture was dissolved in water and further analyzed by HPLC, Purity 80%. 2.7 Analytic Methods The high-performance liquid chromatography (HPLC) system was a ProStar, Model 210 from Varian (Walnut Creek, CA) with a column of Alltima C18LL, 5μm, mm i.d. (Alltech, Deerfield, IL). The mobile phases were (A) water-tfa (100 : 0.025, v/v) and (B) acetonitrile- TFA (100: 0.025, v/v) with a gradient of 0 to 20 % B in A in the first 5 min, 20 to 40 % B in A from 5 to 10 min, 40 to 80 % B in A from min, 80 to 100 % B in A from min, and 100 to 0 % B in A from 20 to 30 min. The flow rate was 1.0 ml/min, and a 10 μl was injected in all analytic experiments. The UV conditions set for detection are two channel modes at 220nm and 254nm. For preparative HPLC, the respective fractions were collected directly from the column according to their retention times. All positive electrospray spectra were acquired using a Bruker Daltonics Esquire 6000 (Bremen, Germany).

47 Synthesis of Metal Nitrosyl Complexes Synthesis of K 2 [RuCl 5 N].xKCl Potassium pentachloronitrosoruthenate(ii) is synthesized as described (Fletcher, J. M., et al., 1955). RuCl 3 nh2 (Ru cont. ca. 41%, Merck) 1.0 g is dissolved in 20 ml of (HCl:H 2 (1:1, v/v), where HCl is 37 % solution of HCl in water) to give a dark brown solution. The solution is refluxed for 1 hr and then evaporated on an evaporating dish by boiling the reaction content at 373 K (a black solid). The black solid is dissolved in HCl:H 2 (1:9, v/v) 20 ml and KN 2 (Baker) ca. 0.3 g is added to the solution. The solution is refluxed for 1 hr. After cooling the solution to room temperature, KN 2 ca. 0.3 g is added to the solution and the solution is refluxed again for 1 hr. ne more addition of KN 2 was carried out and refluxed to give a purple solution. The purple solution is evaporated on a evaporating dish to appear a purple solid. The purple solid is dissolved in HCl:H2 (3:7, v/v) 20 ml and evaporated on an evaporating dish. Finally, the solid dissolved in HCl:H 2 (3:7, v/v) 20 ml with 1.0 g KCl (Baker). The volume of the solution is reduced to ca. 10 ml by evaporation and the solution is cooled to room temperature to give a purple solid. The solid was collected and washed with cold-h2, methanol and ether with yield: ca. 70%. Similarly labeled N complex was synthesized with Na 15 N 2 (ISTEC TM, USA) (Fletcher, J. M., et al., 1955). Quality of the product was analyzed by ESI (m/z, to , Ruthenium isotope distribution) and IR (Appendix 8.3.b) Synthesis of trans-[rucl(n)(cyclam)]cl 2.xH 2 (L4) Equimolar amounts of cyclam (200 mg) and K 2 [RuCl 5 N] (400 mg) were refluxed in 100 ml of methanol for 36 h. After filtration of the reaction mixture through a glass frit, the filtrate was rotary-evaporated to dryness. The resulting brown residue was dissolved in water and loaded onto a cation exchange column. A bluish band that eluted with water was discarded. The yellow product band eluted with 0.2 M HCl. This fraction was vacuum-rotary-evaporated to dryness, and the yellow residue was dissolved in 2:2:1 methanol/ acetone/water solution before it was placed in an chamber for crystallization. Yellow needle-like crystals began to grow within a day. Yield was around 20%. (Lang, D.R., et al., 2000).

48 36 NH N HN 2+ Ru 2Cl - NH Cl HN L Synthesis of trans-[rucln([15]anen 4 )]Cl.xH 2 (RuA) First trans-[rucl 2 ([15]aneN 4 )]Cl complex ([15]aneN 4-1,4,8,12-tetraazacyclopentadecane) was prepared using RuCl 3 3H 2 as starting material, which was obtained from Aldrich. Ruthenium trichloride trihydrate (0.2 g) was dissolved in methanol (50 ml) and refluxed under a hydrogen atmosphere for 4 h. After removing the hydrogen atmosphere, a methanolic solution (25 ml) of 1,4,8,12-tetraazacyclopentadecane (0.2 g, Sigma) was added dropwise to the ruthenium solution under reflux, inert atmosphere, and vigorous stirring. The addition process took ca. 2 h for completion. After further heating under reflux (18 h), the yellowish brown solution was filtered and evaporated to small volume (ca. 10 ml). 1 ml of a saturated aqueous solution of NH 4 Cl (Baker) was added and which resulted in the precipitation of a light yellowish orange solid, which was filtered off and washed with a small volume of methanol and ether. To 0.10 g of trans-[rucl 2 ([15]aneN 4 )]Cl salt dissolved in H 2 (20 ml) was added Zn(Hg) (1.0 g) (Stern) under an inert atmosphere. After 1 h, the ruthenium complex solution was passed through DWEX AG 50W-X2 (Fluka) anion-exchange resin to eluate the products. This procedure was repeated for four times in order to remove free chloride. Nitric oxide was bubbled through that solution (30 min) and the reaction mixture was stirred for another 3 h. That operation was repeated for three times. The volume of the solution was then reduced by rotary evaporation (ca. 10 ml) and 1 ml of a saturated aqueous solution of NH 4 Cl or 12M HCl (5 ml) was added to precipitate the ruthenium complex. The resulting orange precipitate was collected by filtration and washed with diethyl ether and stored under vacuum in the dark. Yield is around 15%, (liveira, F-de-S, et al., 2004) and product was characterized by UV-Vis (Appendix 8.2). This compound is labeled as RuA in cytotoxic studies and complex without nitric oxide is labeled as RuB.

49 37 N NH HN Ru 2+ 2Cl - NH Cl HN RuA 2.9 Biochemical Methods verexpression of Ras wild type, Ras T35S and Ras Y32W in E. coli Expression System All the ptac contructs of the truncated Ras version were selected for overexpression respectively. All the constructs were transformed into E. coli BL21 (DE3) to express the respective proteins. Colonies were selected after transformation on the LB plate with suitable antibiotic. The correct transformants were used to express and purify protein with the following the standard protocols (Tucker, J., et al., 1986). Inoculate 50 ml liquid LB medium containing with suitable antibiotics with the correct E. coli strain and incubate on the shaker at 37 0 C overnight or atleast 12 hours. Transfer the overnight culture to the fresh 10 liters of LB media with suitable antibiotic and incubate at 37 0 C with further shaking. During the 10 liter culture growth, D 600 wass measured at discreet intervals and at D 600 close to 0.6, the culture was induced with 0.1mM IPTG for maximum production of the protein. After induction with IPTG (Fermentas, St. Leon-Rot), the temperature was reduced to 25 0 C, and allowed to grow further 16 hours. For test expression, 1ml of the culture could be transferred to eppendorf tubes for SDS test before IPTG induction and after induction by SDS-PAGE. The overnight culture is centrifuged at 3000 rpm at 4 0 C for 30 minutes. After discarding the spent LB broth, the pellet was washed with PBS solution one more time and centrifuged again. The pellet was dissolved in 2X the weight of the pellet with Buffer A (30 mm Tris-HCl ph 7.8, 5 mm MgCl 2 5 mm DTT (Biomol, Hamburg)), 0.5% PMSF, 0.1mM ATP and all the strains were stored at C respectively until for further use.

50 Purification of Ras wild type, Ras T35S and Ras Y32W proteins from E. coli cultures The respective cell culture pellet which was frozen at 20 C was then thawed under tape water. Thawed cell pellet tube was placed on ice and resuspended in 2.5 ml 1 X phosphate-buffered saline (PBS) solution (140 mm sodium chloride, 2.7 mm potassium chloride, 10 mm sodium hydrogen phosphate, and 1.8 mm potassium dihydrogen phosphate, ph 7.4) or Buffer A. The cell solution was then sonificated for 20 seconds with the maximal power level with amplitude 80. Sonification was repeated with continuous pause and run method on ice for the specified period. After sufficient sonification, the lysed cell solution was transferred to ultracentrifuge tubes, weighed to have an even balance of weight. The tubes were centrifuged at 35,000 rpm for 1 hour at 4 0 C. After centrifugation the clear lysed solution was filtered through 40µm filter, and loaded to the preequilibrated DEAE sepharose column on the AkTA prime by Buffer A. After the loading of the filtered cell lysate, the column was continuously washed with Buffer A, until there is no further unbound protein to column. With a preprogrammed method and in combination of Buffer B (30 mm Tris-HCl ph 7.8, 5 mm MgCl 2 5 mm DTT, 800 mm NaCl), all the bound proteins from the column are eluted. The fractions were analysed with 15% acrylamide gels by SDS-PAGE to identify the regions of the elutant which has maximum content of the Ras protein with minimum contamination of the other forms of the proteins. Care was taken to pool the protein fractions which are separated by maximum molecular weight and with high content of the Ras protein. The pooled protein fractions were concentrated by Amicon concentrator (cutoff 10kDa) and load to preequilibrated gelfiltration column with Buffer C (30 mm Tris-HCl ph 7.0, 5 mm MgCl 2 5 mm DTT, 50 mm NaCl, 0.1 mm GDP). With a preprogrammed method, the proteins were separated according to molecular weights. Higher molecular weight proteins were eluted first followed by lower molecular weight proteins. The fractions were analyzed with 15% acrylamide gels by SDS-PAGE in comparison to marker protein and standard Ras protein which has molecular weigth of 18.5 kda. The identified Ras protein was pooled respectively and its concentration was estimated by Braford assay (Braford, M.M., 1976).

51 Nucleotide exchange Nucleotide exchange was performed according to (Tucker, J., et al., 1986) EDTA method or Alkaline Phosphatase (AP) method. In the first method by EDTA, this chelates the Mg 2+ ions leading to an increase in the dissociation rate of the nucleotide. The bound nucleotide can then be exchanged by an excess of freshly added nucleotide. A master solution containing 100 mm EDTA, 150 mm ammonium sulfate and 10 mm nucleotide (stock 100 mm nucleotide in 1 M Hepes, ph 7.5) was made in 50 mm HEPES (ph 7.5), 100 mm NaCl and 5 mm DTT. This is incubated for 60 minutes at room temperature or overnight at 4 C with 300µl of 5 mg/ml of the protein. The exchange reaction was stopped by adding 150 mm MgCl 2. Non-bound nucleotide was removed by washing the protein several times with an Amicon concentrator (10 kd cutoff) at 4 C followed by gelfiltration by NAP-5 column. To confirm successful nucleotide exchange, the nucleotide concentration was determined by HPLC (Section 2.9.4). The protein was flash frozen and stored at -80 C. In the second method the exchange to caged-gtp was done according to a modified protocol of (John, J., et al., 1988). Ras GDP was incubated with a fivefold excess of caged-gtp and catalytic amount of alkaline phosphatase in a buffer containing 50 mm Tris (ph 8.5), 5 mm DTT, 10 µm ZnS 4 and 200 mm ammonium sulfate. After 3 hours of incubation at room temperature, the protein was rebuffered and excess caged nucleotide was removed by gel-filtration using NAP-5. The fractions were analyzed by HPLC (Section 2.9.4). Typically, the exchange to caged-gtp was approximately 60% complete, the rest being GDP. The exchange process was good in the entire attempt with NPE GTP, while php GTP conversion sometimes was not observed with good nucleotide exchange Nucleotide detection using reversed-phase HPLC The principle of nucleotide separation is the interaction between the hydrophobic static phase and the ion pair of nucleotide and tetrabutylammonium in the mobile phase. Depending on the number of phosphates, a variable number of tetrabutylammonium ions are bound by the nucleotide, which increases the retention time on the column. The sample was applied on a HPLC system Gold 166 (Beckman, Palo Alto, USA) and separated via a reversed-phase column DS Hypersil C18 (Bischoff, Leonberg). Denatured proteins were adsorbed at a nucleosil-100- C18 precolumn. The running buffer contained 10 mm tetrabutylammoniumbromide, 100 mm potassium phosphate (ph 6,5) with 7,5% to 15% acetonitrile. Nucleotide peaks were detected by

52 40 measuring adsorption at 254 nm and quantified by integration. The column was calibrated by standard nucleotide solutions Time resolved infrared spectroscopy Instrumentation A typical time resolved FTIR instrument contains a Globar (SiC, silicon carbide) (light source at 1500 K). Infrared light from the globar passes an aperture (0.25 mm to 12 mm) before entering a Michelson interferometer (Figure 2.1.a). Subsequently the light passes through sample chamber, which is equipped with a thermostatic transmission cell in which the sample is placed. Finally the infrared light reaches a liquid nitrogen cooled MCT (mercury-cadmium-tellurium) detector. For triggering of a reaction, the sample in the cell can additionally be irradiated by a UV-laser. An FTIR instrument has crucial advantages over a dispersive spectrometer. By means of the incorporated Michelson interferometer (Figure 2.1.a), all wavelengths can be measured in parallel (multiplex / Felgett advantage). At the beam splitter, one half of the infrared light is reflected onto a fixed mirror, while the other half is transmitted to a moving mirror. Both parts then recombine at the beam splitter. Depending on the position of the moving mirror, a path difference ( x) is created between the beam reflected by the fixed mirror and the moving mirror. The moving mirror at position x1 will lead to a path difference of zero between two waves of a monochromatic radiation and therefore to an intensity increase by a factor of 2, whereas a path difference of half a wavelength (mirror position x2) leads to extinction. This phenomenon is known as interference. Therefore mentioned cases are known as constructive and destructive interference, respectively. For monochromatic radiation, the Interferogram can be described as a cosine function of the mirror position x as shown by equation 2.1. I 0 designates the partial radiation intensity. Polychromatic light source therefore leads to an overlap of several cosine functions. If a polychromatic radiation is used, the individual contributions are to be integrated as described by equation 2.2 below.

53 41 In this way, variation of path difference alters the interference pattern after the recombination, producing an interferogram in which intensity is plotted against mirror position x (Figure 2.1 b, Interferogram). Through mathematical operation known as Fourier transformation (equation 2.3), a single channel spectrum (Figure 2.1 b) is obtained, where the intensity, I, is a function of the wavelength. Figure 2.1 Instrumental Setup: (a) Schematic representation of a Michelson interferometer. An electromagnetic wave is splitted at the beam splitter. (b) The result of measurement of an interferogram, where intensity is plotted against the mirror position. After Fourier transformation, the intensity (I) is obtained as a function of the wavelength (single channel spectrum) [modified from (Kötting, C., et al., 2005)]. An absorbance spectrum is obtained by comparison of two single channel spectra, one with sample and one without sample respectively. As an example, absorbance spectra (A) of a protein can be calculated from the single channel spectrum of the protein (I) and the single channel spectrum of the buffer as reference (I 0 ) by equation 2.4.

54 42 In practice, the Interferograms are not recorded continuously, but in n discreet points n x with the maximum path difference of the partial beams N x. Hence interferogram consists of N discreet points at an interval of x. The spectral resolution is the reciprocal of the product from the point distance x and the number of points N as described in equation 2.5. The calculation of the spectrum from the discreet interferogram can be done by discreet Fourier transformation as described by equation 2.6. With modern spectrometers, a complete spectrum can be obtained within 10 milliseconds. Further advantages of FTIR-spectrometers are the absence of dispersive elements (slits combined with prisms or gratings which attenuate the signal intensity: Jaquinot advantage), and the high accuracy of the wavelength (Connes advantage) Basic principles of Infrared spectroscopy Any molecule, including proteins can be envisioned as a system of mass points connected by springs (the bonds). Such systems can undergo distinct vibrations. A vibration can be induced by electromagnetic field, provided the energy of the field matches to the energy of the vibrational mode and the dipole moment of the molecule changes during this vibration. These interactions result in absorbance of distinct quantized energies. In case of vibrational changes, such energies are in the infrared spectral range. Therefore, infrared spectroscopy is a vibrational spectroscopy. The stretching frequency ν can be correlated to the reduced mass (µ) and force constant (k) as described by equation 2.7.

55 43 If one of the atoms of such a pair is replaced by an isotope, the force constant does not change significantly but the reduced mass (µ) changes. The isotopic stretching frequency ν isotope can be easily calculated by the following equation 2.8. This is the basis of infrared band assignment which can be done either by isotopically labeled protein (Engelhard, M., et al., 1985) or in case of nucleotides, by specific 18 labeling (Allin, C., et al., 2001a,b) or by amino acid exchange via site directed mutagenesis (Gerwert, K., et al., 1989). Even a relatively small protein of 20 kd (like Ras) contains about 104 vibrational modes. In addition, hydration is necessary for activity of biomolecules. Since water is a strong absorbent in the infrared region, from a typical absorbance spectrum one can obtain global information of proteins but not about small individual changes. This problem is circumvented by difference spectroscopy where two absorbance spectrums are subtracted from each other. Typically, for a reaction A B, one calculates (B A). Thus, the vibrations of groups, which are unchanged during the reaction, are cancelled out and only the ones that change during the reaction are seen. In this process, individual absorbances of groups can be resolved from the background, which are 10 3 weaker. This is possible through identical measurement conditions, high sensitivity and stable instrumentation Sample preparation Figure 2.2 shows a typical transmission cell with IR transparent windows (e.g. CaF 2 or BaF 2 ). Due to high absorptivity of water in the mid infrared spectral region, meaningful spectra of hydrated proteins are obtained only by transmission measurements through very thin film (2 10 µm), adjusted by a mylar-spacer. Small aliquots of protein solution is placed on the IRtransparent window and carefully concentrated under a nitrogen stream. A typical measurement requires about µg protein and the concentration of protein in the film is 6 10 mm. The sample chamber is closed by a second IR-window and sealed by means of the -rings. The sample holder has high thermal conductivity and it is placed inside the spectrometer for thermal equilibration (Allin, C., et al., 2001a,b). The sample solutions for the measurements with Ras-

56 44 caged nucleotides are conducted in buffer with 100 mm HEPES (ph 7.5), mm MgCl 2, 10 mm DTT and 12% ethylene glycol. DTT was added to scavenge the reactive photolysis byproduct 2-nitrosoacetophenone (Allin, C., et al., 2001a,b). All through the experiments, the concentration of the ligands were equated with respect to concentration of the Ras protein, whenever described in the results. For nitrosation experiments, the buffer and reaction mixture of the Ras protein was without any DTT. The selection of caged nucleotide depends on type of the experiment. For instance, in T35S and RalGDS interactions phpgtp was used, while intrinsic GTPase activity was measuremed with NPEGTP. Figure 2.2 Schematic representation of a transmission cell, protein sample is held between two windows (CaF 2 /BaF 2 ) in an air tight fashion (Kötting, C., et al, 2005) Initiation of the reactions and data collection After sufficient stabilization of the sample which is indicated by stable difference single channel measurement, the reaction is initiated by a sharp UV-Laser trigger. In the context of GTPase reactions this is achieved by photo labile cage compounds as shown in Figure 2.3. The 1-(2- nitrophenyl) ethyl (NPE; Figure 2.3 a) moiety is frequently used to protect phosphate, nucleotides and nucleotide analogues. Application of UV flashes leads to photolysis, forming GTP and the by-product 2-nitrosoacetophenone (Figure 2.3 a) (McCray, J.A., et al., 1980). A spectrum of caged GTP is measured prior to the photolysis as reference. After photolysis further spectra are recorded (Figure 2.3 c, A to B part) and absorbance amplitude spectra are calculated

57 45 (Figure 2.3 d, A to B part) through global fit (section ). For a GNBP bound to caged GTP and even with another protein like GAP, the basic principle is the same, as shown in Figure 2.3 b. In this case, the reaction proceeds to hydrolysis, with the involvement of intermediates in case-to-case basis. The spectra recorded in this work were collected in rapid scan mode. The principle of rapid scan FTIR method is: after taking a reference spectrum of the sample in its ground state, it is activated (e.g. by a laser flash) and interferograms are recorded in much shorter time than the half lives of the reactions (Gerwert, K., et al., 1990). The first four reference (R) interferograms (Figure 2.3 c) represent ground state (A) (Figure 2.4 d) while the following interferograms are taken during the reaction pathway (B to C to D). Velocity of the interferometer s moving mirror, V max, and the desired spectral resolution ν determines the scan duration t (time resolution) as described by equation Measurement parameters Sample composition: Protein solutions were concentrated to a final concentration of 6-10 mm on 20x2 mm CaF 2 or BaF 2 windows under nitrogen stream along with selected buffer system. Typical buffer composition is: 200 mm Buffer (HEPES for ph 7.6), 20 mm DTE / DTT, 20 mm MgCl 2. Intrinsic experiments were conducted at 293 K and 303 K, while 1% GAP catalyzed reactions was done at 283 K and photolysis experiments were done at 258 K, along with 12% ethylene glycol to avoid freezing. Special instruments: IFS 66v/s FTIR spectrometer (Bruker ptics, Karlsruhe, Germany) with KBr beamsplitter, MCT detector KMPV11 1 J1 (Kolmar, Newburyport, MA, USA), Excimer-Laser LPX 240i (Lambda Physik, Göttingen, Germany) with XeCl (308 nm), CaF 2 / BaF 2 windows (Korth GmbH, Kiel, Germany), Mylar film (DuPont, Circleville, H, USA).

58 46 Figure 2.3 Photolysis scheme (a) Photolysis of caged [1-(2-nitrophenyl) ethyl] GTP. (b) Time course of a rapid scan FTIR experiment, investigating the interaction of two proteins (in red and blue color). (c) Time course of data acquisition. First reference spectra (R) are taken. Then a laser flash initiates the reaction. During the reaction via intermediate C through product D, interferograms are continuously recorded. (d) After kinetic analysis, e.g. by global fit amplitude spectra are obtained, showing the bands of the groups involved in the reaction. [Modified from (Kötting, C., et al., 2005)]. perational mode: The intrinsic reactions were monitored in double sided forward backward mode with a scanner velocity of 100 KHz and the GAP catalyzed reactions and photolysis experiments were monitored in double sided forward backward mode with a scanner velocity of 320 KHz. In both cases, the spectral resolution was 4 cm 1 and folding limit cm 1. For nitrosation experiments, 4000 cm -1 cutoff filter was used, while 1900 cm -1 cutoff filter was used for other experiments. The laser energy was between 90 and 120 mj per flash with pulse duration of ~ 20 ns. Numbers of flashes were varied between 20 and 60 depending upon the type of the caged nucleotide. The chosen number of flashes was a compromise between the time of photolysis and the degree of conversion. A time window of 14 ms per interferogram was allowed for the scanner to return to initial position while measuring at 320 KHz. The time

59 47 resolved data were collected by PUS TM (version 5.0, Windows 2000, XP) and analyzed by global fit programs with riginlab (USA) and Matlab 12.1 (MathWorks, Aachen, Germany) Data analysis Difference spectra for the 1% GAP catalyzed reaction (or intrinsic reaction) were made by using the software PUS TM (Bruker). The data of the time-resolved fast scan measurements for the GAP catalyzed reactions were analyzed between 1800 and 950 cm 1 with a global fit method (Hessling, B., et al., 1993). The global fit analysis not only fits the absorbance change at a specific wave number, but also upto 800 wave numbers in the spectrum simultaneously. All reactions are assumed to be first order and can therefore be described as a sum of exponentials. The fit procedure minimizes the difference between the measured data A and the theoretical description A, weighted according to the noise w ij at the respective wave numbers, and summarized not only over time (t j ) but also the wave numbers (i). Thus, the absorbance changes A in the infrared are analyzed with sums of n r exponentials with apparent rate constants k l and amplitudes a l as described in equation In the analysis, the weighted sum of squared differences (f) between the fit with n r rate constants (k l ) and data points at n w measured wavenumbers (ν i ) and n t time-points (t j ) is minimized as shown in equation For unidirectional forward reactions, the determined apparent rate constants are directly related to the respective intrinsic rate constants describing elementary reaction steps. If significant back reaction also occurs, the analysis becomes more complicated. In this case, the reaction has to be modeled until the estimated intrinsic rate constants fulfill the experimentally observed time courses described by the apparent rate constants. Because the number of intrinsic rate

60 48 constants needed in a model is usually larger than the experimentally observed ones, the problem is experimentally under determined, and the solution is equivocal. This limitation holds for any kinetic analysis, not only for IR measurements. The very slow intrinsic reaction of GTPase was difficult to monitor due to baseline drifts and kinetics are measured by the difference in adsorption of the α-gtp and α-gdp, the resulting differences are plotted with respective to time. Kinetics is calculated from the plot with a single exponent first order equation (Kötting, C., et al., 2004). The representation and annotation of the experimental data is followed according to the Scheme 3, here for phpgtp, k 0 (A B) represents the photolysis spectra with the generation of Ras ff GTP state, while k 1 (B C) represents the conformation change between Ras 0ff GTP state and Ras n GTP state shown with amplitude spectra, finally k 0 + k 1 (A C) represents the photolysis difference spectrum. Scheme 3. Decaging reaction of NPEcagedGTP and phpgtp hv Ras*cgGTP Ras*aci-nitro anion Ras*GTP Ras*GDP + P i H + alpha NAP A B C D Photolysis difference spectrum Photolysis amplitude spectrum Hydrolysis difference spectrum hv Ras*phpGTP Ras off *GTP Ras on *GTP Ras*GDP + P i k 0 k 1 A B C D Photolysis difference spectrum Amplitude spectrum (Conformation Change) Hydrolysis difference spectrum n the other hand, for the NPEcagedGTP, only k 0 + k 1 is observed, which is the photolysis difference spectra, k 0 is not resolved due to slow decomposition of the aci-nitro anion. From the global fit analysis, (C D) represents hydrolysis amplitude spectrum which was previously described as hydrolysis difference spectrum (Allin, C., et al., 2001a,b)

61 Ab initio calculations on triphosphate models The fully optimized geometries of methyl triphosphate (T1, T2, T3) were obtained by the analytical gradient methods using the basis set G** (Petersson, G.A, et al., 1988, Hansia, P., et al, 2006). The protonated γ phosphate model was adapted as described by Hansia, P., et al., The calculations were performed using Gaussian03 (Gaussian 03, 2004) program at the level of hybrid Density Functional Theory (B3LYP) (Becke, A.D, 1993). The default fine integration grid (corresponding to Int=FineGrid) and the default convergence criteria (Maximum Force threshold = Hartrees/Bohr, RMS Force threshold = Hartrees/Bohr, Maximum Displacement threshold = Bohr and RMS Displacement threshold = Bohr) were used for DFT calculations (B3LYP) (Hansia, P., et al., 2006). Furthermore, the optimized geometries were used to carry out calculations on vibrational frequencies on the different models by DFT level of theory (B3LYP) with keyword Freq and electrostatic potential (ESP) charges are calculated with keyword MK. The frequency modes were identified from GaussView. - P - P T1 - P H - P - P T2 H N - P H - P - P T3 H 2 C - P H - P P X P H - - C1 C2 Figure 2.4 Model compounds of triphosphate, T1, T2 and T3 are representative models of the GTP, GppNHp and GppCH 2 p respectively. C1 represents the distance between α phosphate and β phosphate, while C2 represents the distance between β phosphate and γ phosphate and X is heteroatom,, NH or CH 2. The main emphasis of the calculations were also directed at the probing of the variation in the distances between each phosphate centers calculated as C1 (α-, & β-phosphate), C2 (β-, & γ- Phosphate) with respect to bridging atom X, here X is, NH, CH 2. Comparison was made

62 50 between C1 and C2 from the model systems to the crystal structures of the proteins with bound nucleotides (Figure 2.4, Appendix 8.4) Nucleotide dissociation with metal nitrosyl and peroxo complexes The nucleotide dissociation from the Ras protein was carried with addition of 10 times excess of the K 2 [RuNCl 5 ] and Titanium peroxocitrate with freshly prepared DTT free Ras*GppNHp(1mM) or Ras*GDP(2mM). In either case excess of the GDP was added to the reaction mixture and analysed by HPLC (Heo, J., et al., 2004). The whole content (250 µl) was then transferred to an UV cuvette. The reaction mixture in the UV cuvette was exposed to UV irradiation at 360 nm for 15 minutes. After the UV irradiation, the reaction mixture was centrifuged; the solution was analyzed by HPLC. The flowthrough was also analyzed by HPLC and ESI after applying the reaction mixture to the Amicon concentrator (10 kda cutoff) Fluorescence spectroscopy / MantGDP-exchange assay All experiments were conducted at 25 C in HEPES-Mg-buffer (20 mm HEPES ph 7.4, 5 mm MgCl 2, 150mM NaCl). Ras*MantGDP protein final concentration was 0.1 µm, 1,2,3-hexanetriol and GDP were 200 and 100 μm respectively. Measurements were performed in Perkin Elmer instrument with a 750µl quartz cuvette. Mant-fluorophore was excited at 366 nm and emission was collected at 450 nm; Nucleotide exchange reactions were started by addition of 50 μm of 1, 2, 3-Hexanetriol (final concentration). Measurements were conducted for at least 600 seconds. Rates were determined by monoexponential curve fitting (y = y o + ae -kt ) using the program rigin (riginlab, USA) Cytotoxicity experiments Cell culture conditions The human HT29 colon cancer cell line was obtained from the Institute of Pharmacy of the Free University of Berlin. The Cell line was cultured in McCoy Medium (Sigma, Germany) supplemented with 10% Fetal Calf Serum (Gibco, Germany), 2 mm L-Glutamine, 100 U/ml Penicillin and 100 µg/ml Streptomycin. Cells were incubated in 75 cm 2 cell culture flasks at

63 51 37 C and in a humidified atmosphere with 5% C 2. The cell lines were transferred weekly using 0.05% Trypsin with 0.02% EDTA (Gibco, Germany). Cells were treated the following day. Serum-free medium was used during treatment with test compound Cytotoxicity Assays The crystal violet assay (Bernhardt, G., et al., 1992) and the resazurin assay ('Brien, J., et al., 2000) (Sigma) were performed with the HT29 cell lines on 96-well micro titer plates. 100 µl of a cells/ml suspension of culture medium were plated into each well and incubated for 24h at 37 C and 5% C 2. By addition of one µl of a stock solution of the respective compound dissolved in methanol or water until the desired concentration is reached. After 48h hour of incubation of the HT29 colon cancer cells in the presence of the compounds, the cells were irradiated with UV light at 365 nm for a period of 10 and 25 minutes respectively. The cells were incubated further one more day and assayed by the resazurin and then the crystal violet assay. Cells were washed two times with colourless RPMI 1640 medium (PAA Laboratories, Germany) without Phenol red and without Fetal Calf Serum. After that 90 µl of colourless RPMI 1640 and 10 µl of resazurin were added to each well. Absorbance was directly measured at 600 nm, the measurement before irradiation of the compound with UV and was also repeated after two hours of incubation with RPMI 1640 medium. Activity of mitochondrial dehydrogenases was determined as decrease in Absorbance. After that the cell biomass was determined by a crystal violet assay. The medium was removed and cells were fixed with 4% Paraformaldehyde in PBS. Cells were washed with PBS (phosphate buffered saline) and afterwards with 0.1% Triton-100 (Sigma) in PBS. Cells were then stained with a 0.04 % crystal violet solution and subsequently washed four times with distilled H 2. Crystal violet was extracted by 96% Ethanol and absorbance was determined at 570 nm. Values were corrected by absorbance at start of substance incubation. Vehicle controls (methanol), negative controls (culture medium) and positive controls (Cis-Platin (Sigma)) were also determined.

64 52 3 Results 3.1 Studies on Semisynthetic Ras ff to n conformation of the semi-synthetic Ras protein Results are classified into two parts according to the type of the semi-synthetic Ras protein provided from MPI, Dortmund (Appendix 8.5). ff to n conformations discussed in this part comprise only from the semisynthetic protein as shown in the Appendix 8.5.a. The results indicate the fraction of the semi-synthetic Ras protein which is confirmed from SDS-PAGE (Appendix 8.5.a) has a similar but not the same photolysis behavior like wild type (Figure 3.1.a). The wild type shows the characteristic peaks of γ-phosphate vibrations at 1145 cm -1 and β-phosphate vibrations at 1214 cm -1 in the ff state formed immediately after the photolysis of the caged phpgtp. For the semisynthetic protein, the γ-phosphate vibrations are observed at 1154 cm -1 instead of 1145 cm -1 with a band difference of 9 cm -1. The amide regions are different for the initial ff state conformation, where threonine 35 residue has no coordination to γ- phosphate and Mg 2+ (not shown in the Figure 3.1.a). The initial state of the protein immediately after photolysis undergoes conformation changes to attain a final n state conformation. In the amide I region, for the semisynthetic protein there is a decrease in the intensity compared to wild type. The intensities around the phosphate regions are also observed with some changes in semisynthetic Ras protein; with decreased intensities of the γ-phosphate vibrations. There is a small blue shift of 4 cm -1 for γ-phosphate, which is observed at 1148 cm -1 compared to wild type which has the characteristic γ-phosphate vibration at 1144 cm -1. The amide I vibrations of Threonine (Thr35) at 1689 cm -1 are similar in both cases. The final n state of the protein after it attains a stable conformation is shown in the Figure 3.1.b, is described by the photolysis difference spectra, which is the difference between the final state after conformation change attained with no further changes occuring in any portion of the protein and the ff state related to the protein caged state before photolysis. The amplitude spectra corresponding to the difference between Ras on GTP and Ras off GTP for the semisynthetic and normal Ras protein are shown in the Figure 3.1.c, positive peaks are due to the Ras on GTP state, and negative peaks from the Ras off GTP state.

65 53 Figure 3.1.a Semisynthetic Ras protein characterization Comparison of the ff states of wild type Ras protein (Black) and semisynthetic Ras protein (Red) immediately after cleavage of the p-hydroxylphenacyl moiety from caged phpgtp and before any conformational changes, in HEPES, ph 7.5, 258 K representing rate k 0, scaled between cm -1. Figure 3.1.b Semisynthetic Ras protein characterization: Photolysis difference spectra of Ras wildtype (Black) and Ras semisynthetic protein (Red), representing rate k 0 + k 1, scaled between cm -1.

66 54 For semisynthetic Ras protein only minor changes are observed in the amplitude spectra. This spectrum represents the overall changes associated with transition from ff state to n state. The main differences in the semi-synthetic Ras protein are a small blue shift in the γ-phosphate vibrations to 1144 cm -1, and also decrease in the intensities of the γ-phosphate vibration and amide I regions around cm -1. The kinetic behavior of the transition from ff to n state of the Ras wild type and Ras semisynthetic protein is shown in the Figure 3.1.d. The kinetic behavior of the Ras wild type and Ras semisynthetic protein transition from ff to n state is described with a simple first order exponential. The rate ff-n of the transition for the semisynthetic Ras protein is 8 s -1, while Ras wild type folded to n state at 6 s -1. Figure 3.1.c Semisynthetic Ras protein characterization: Amplitude spectra representing the difference between Ras on GTP and Ras off GTP. Positive peaks are due to the Ras on GTP state, negative peaks are from the Ras off GTP state. The spectra shown in the Black is the Ras wildtype while the spectra in the Red is from semisynthetic Ras protein 1-166, representing rate k 1.

67 55 Figure 3.1.d Semisynthetic Switching Kinetics of γ phosphate, 1144 cm -1 : Kinetics of the switching from ff state to n state. Ras Wildtype protein is shown in the Black (6 s -1 ), while semisynthetic Ras protein is shown in the Red (8 s -1 ). The kinetics is fitted to simple first order exponential, and measurement was conducted in HEPES, ph 7.5, 258 K NF1 activation of the semisynthetic Ras protein The influence of 1:1 complex formation of the NF1 with semisynthetic Ras protein is compared with Ras wildtype and also to semisynthetic protein without NF1 (Figure 3.2.a). The changes on association of NF1 with semisynthetic Ras are similar to semisynthetic Ras protein without any NF1, only differences were observed in the amide region intensities around 1655 cm -1, γ- phosphate intensity and downshift in the β phosphate vibrations. In the presence of NF1, semisynthetic Ras protein showed a blue shift in the amide region at 1659 cm -1, while without any NF1, semisynthetic Ras protein showed the same peak at 1655 cm -1 with diminished intensity which was also observed for wild type protein. In the β-phosphate regions, in comparison to wild type which is indicated by a peak at 1217 cm -1, semisynthetic Ras protein on association with NF1 is observed with a red shift of 5 cm -1 resulting a peak of β-phosphate at 1212 cm -1, while without any NF1 semisynthetic Ras protein showed the β-phosphate vibration

68 56 at 1214 cm -1. In the case of the γ-phosphate vibration, for semisynthetic Ras protein, a peak was observed at 1147 cm -1 with a blueshift of 4 cm -1 on association with NF1 compared to wild type, while without any NF1, semisynthetic Ras protein still showed blue shift of 5 cm -1 resulting a peak at 1148 cm -1. Figure 3.2.a Interaction of Semi synthetic Ras with NF1. Photolysis difference spectra of Ras wildtype (Black) and Ras semisynthetic protein (Blue), Ras semisynthetic protein with NF1 (Red, dotted). The semisynthetic Ras protein fractions from the Appendix 8.5.b were also analyzed similarly. All the fractions of the semi-synthetic Ras protein were compared with respect to the photolysis activity (Figure 3.2.b) and also hydrolysis activity, where hydrolysis rates can be obtained by the difference of the absorption at 1236 and 1262 cm -1 of the α phosphates of GDP and GTP respectively, hydrolysis rates are shown in the Figure 3.2.c. The rate corresponding to 5% NF1 activation of Ras*GTP hydrolysis was sec -1 which is shown in the Figure 3.2.c (Black line), while none of the semisynthetic Ras protein fractions showed any hydrolysis either in FTIR measurements (Figure 3.2.b) or in the SDS PAGE (Appendix 8.5.b) or GAP assisted GTPase activity (Figure 3.2.c).

69 57 Figure 3.2.b Interaction of Semi synthetic Ras with NF1: Comparison of photolysis difference spectra between Ras wild type (Black) and all fractions of the semisynthetic protein with 1:1 complex of NF1 (Green). Absorbance Difference ( ) log 10 time (sec) Figure 3.2.c GTPase activity of the semisynthetic Ras protein. Kinetics of the Ras GTPase activity is calculated by plotting the absorbance differences between α-gdp and α-gtp with respect to time, calculated rates are from a simple first order exponential fitting. Data corresponding to wildtype is shown in red with 5% NF1, while all the remaining data are from semisynthetic Ras protein with 1:1 complex of NF1. Measurements were carried in MES, ph 6.0, 283 K.

70 Modulation of Ras protein by Zinc cyclen Experiments were done based on the prediction of modulation of the Ras Effector interactions with zinc cyclen (Spoerner, M., et al., 2005) Interaction of Zinc cyclen with Ras protein Results indicate that in the presence of ten (10X) and twenty (20X) times excess of the Zinc cyclen comparatively to Ras protein concentration, Ras protein has no major changes in the kinetics of the switching from ff state to n state as shown in the Figure 3.3.a. Figure 3.3.a Rate of switching of Ras protein in the presence of zinc cyclen: The plot is the generation of the γ phosphate vibration at 1144 cm -1 of the Ras on GTP in the presence of the zinc cyclen at 10X (Blue, 6.3 s -1 ) and 20 X (Red, 6.1 s -1 ) concentration compared to that of the Ras protein at 258 K. Control Ras protein without Zinc cyclen is shown in black (6.2 s -1 ). Measurements were done in HEPES, ph 7.5, 258 K.

71 59 The rate of the transition from ff state to n state was observed at a range of 6 s -1 in all cases with only very minor variations in the presence of Zinc cyclen (Figure 3.3.a). The amplitude spectra representing the conformation changes from ff state to n state was comparable to that of Ras protein without any zinc cyclen complex (Figure 3.3.b). The amplitude spectra of the Ras*GTP in the presence of 10X and 20X of zinc cyclen are comparable with slight differences in the intensities of the γ-phosphate at 1142 cm -1 (Figure 3.3.b). In the presence of the Zinc cyclen, the intensity of the γ-phosphate was decreased by 10 percent without any red or blue shift of the peak. The other characteristic peaks of the amplitude spectra representing the conformation changes from ff state to n state are also represented at 1689, 1262, and 1220 cm -1 depicting the vibrations of the Threonine 35 backbone, α-phosphate and β-phosphate vibration respectively (Figure 3.3.b). Some minor changes were observed at other regions which are at present unassigned, and it is not the scope of this study to speculate its origin around 1410 and 1090 cm -1. Figure 3.3.b Interaction of Ras*GTP protein with Zinc cyclen: amplitude spectra representing the conformational changes associated with interaction of Zinc cyclen with Ras*GTP, Ras wild type, control (Black), in the presence of 10X of Zinc cyclen (Blue), in the presence of 20X of Zinc cyclen (Red), scaling is done at 1689 cm -1 representing the rate k 1.

72 60 The final equilibrium state after the interaction of Zinc cyclen with Ras*GTP is shown in the photolysis difference spectra (Figure 3.3.c), the results indicate in the presence of the 20X of Zinc cyclen, the intensity of the γ phosphate was reduced around %. n the other hand, the β-phosphate vibration in the presence of 20X of Zinc cyclen was observed at 1219 cm -1. The other differences might be due to slight precipitation of the protein due to the presence of high concentration of the Zinc cyclen (20X), while the characteristic peak of the backbone vibration from the Threonine 35 is unperturbed at 1689 cm -1. Figure 3.3.c Interaction of Ras*GTP protein with Zinc cyclen: Photolysis difference spectra of Ras protein in the presence of Zinc cyclen at 10X (Blue) and 20X (Red) times the Ras protein concentration at 258 K, php caged GTP was used, and scaling was done between cm -1 representing the rate k 0 + k 1, Control is shown in the Black. The combined results from the Figure 3.3.a, b, & c indicate that in the presence of Zinc cyclen there is no effect on the natural orientation of the phosphate groups or the switch regions of the Ras protein which is contrary to what was speculated or proposed by Spoerner, M., et al., 2005, which were conducted on Ras*GppNHp instead of Ras*GTP. The present study represents that conformations of the Ras protein in GTP bound state is unique and not perturbed by any phosphate chelating agents like Zinc cyclen.

73 Interaction of Zinc cyclen with ff state Ras mutants Further experiments were conducted similar to Ras protein on the ff State mutants like T35S and Y32W proteins in the presence of the RafRBD. The basic idea was to probe the nature of the differences in switch regions in the presence of a chemical compound which has a tendency to act as a phosphate chelate and also to answer questions whether RafRBD still has the ability to interact with switch regions of GTPases in the presence of the chemical compounds. Results on Y32W protein without interaction with RafRBD protein show orientation of the Y32W is perturbed in the presence of zinc cyclen with a red shift of 8 cm -1 for the β-phosphate vibration (Figure 3.4.a, Red), which indicate that zinc cyclen has a tendency to interact with phosphate moieties if the switch regions are open. In the presence of the RafRBD, the native property of the switching of the protein from ff state to n state is observed with restoration of the β phosphate vibration to 1214 cm -1. Figure 3.4.a Photolysis difference spectra of Y32W in complex with RafRBD and Zinc cyclen: Y32W, Raf RBD and Zinc cyclen (1:1:5) (Black), Y32W and RafRBD (1:1) (Green), Y32W (Blue), Y32W and Zinc cyclen (1:5) (Red). Measurements were carried at 258 K in HEPES, ph 7.5. NPE caged GTP was used, scaling is done at 1345 cm -1.

74 62 The results demonstrate the role of RafRBD to identify the ubiquitous switch region even in the presence of a phosphate chelate agent like zinc cyclen. The intensities of the γ-phosphate vibrations are increased in the presence of RafRBD and zinc cyclen (Figure 3.4.a, Black). Switching on the other ff state mutant like T35S protein was also conducted similarly. The difference between T35S and Y32W is the orientation of the Threonine 35 residue, in which T35S protein lacks the coordination of the threonine to Mg 2+ and γ phosphate in the ground state in the absence of the RafRBD. In the case of Y32W, the switch regions might be in an unknown open conformation even after coordination of the Threonine 35 to the γ-phosphate, while for the ff state in T35S protein, there is no coordination of the serine residue which is present instead of threonine at the position 35. Results indicate in the presence of the zinc cyclen (10X), the T35S protein has a tendency to recognize RafRBD, i.e., it shows similar behavior as compared to the protein interaction between RafRBD and T35S without any compounds. The minor differences are in the regions of the β-phosphate, which is blue shifted to 1225 cm -1 instead of 1217 cm -1 (Figure 3.4.b). Results also indicate that in the presence of Zinc cyclen, T35S protein exists in high percentage of n state conformation upon interaction with RafRBD. Figure 3.4.b Photolysis difference spectra of T35S in complex with RafRBD and Zinc cyclen: T35S, Raf RBD and Zinc cyclen (Red, 10X), T35S and RafRBD (Black), measurements were done in HEPES, ph 7.5, 258 K with phpgtp, scaling was done between cm -1.

75 k = 5.5 sec -1 k = 4.5 sec -1 n State Δ A? T35S + RafRBD (1:1) T35S + RafRBD + Zinc Cyclen (1:1:10) log 10 time (sec) ff State Figure 3.4.c Switching kinetics of T35S in the presence of Zinc cyclen (10X) and Raf RBD at normalized 1144 cm -1 intensity: T35S, RafRBD and Zinc cyclen (5.5 sec -1, Red, 10 X) T35S and RafRBD (4.5 sec -1, Black), measurements were done in HEPES, ph 7.5, 258 K. H H H H H H Lupeol Ileal Lipid binding protein Figure 3.4.d Schematic representation of Ileal Lipid binding protein (Kouvatsos, N., et al., 2006) and Lupeol.

76 64 Δ A k = 5.3 sec -1 k = 4.5 sec -1 T35S + RafRBD (1:1) T35S + RafRBD + Lupeol (1:1:0.2) ff State log 10 time (sec) n State Figure 3.4.e Switching kinetics of T35S in the presence of Lupeol (0.2X) and Raf RBD at normalized 1144 cm -1 intensity: T35S, RafRBD and Lupeol (5.3 sec -1, Blue, 0.2 X) T35S and RafRBD (4.5 sec -1, Black), measurements were done in HEPES, ph 7.5, 258 K. The rate of switching from ff state to n state in the presence of Zinc cyclen (10X) for the protein complex of T35S and RafRBD is shown in the Figure 3.4.c. Rate observed close to 5 s -1 indicates a normal behavior of the protein interactions between the T35S and RafRBD in the presence and also in the absence of the Zinc cyclen, demonstrating that Zinc cyclen has no effect on the protein interface recognition between T35S and RafRBD similar to Y32W protein and RafRBD. Experiments were further conducted with Lupeol, which is known to bind a common Ileal lipid binding protein, the protein which has the conserved domain structure similar to ubiquitin (Kouvatsos, N., et al., 2006, Figure 3.4.d), though at present there are no crystal structures of this ligand binding to the domain of this protein. In the present study, Lupeol has lower solubility in the water; only 0.2X of Lupeol experiments were conducted compared to concentration of the T35S protein. Results indicate this form of ligand (Lupeol) has no role in modulation of the RafRBD interaction with T35S (Figure 3.4.e), even though in cell assays this molecule is shown to inhibit cell growth and promotion of apoptosis (Lee, T.K., et al., 2007). The rate of switching

77 65 in the presence of the zinc cyclen and lupeol were very close to the normal values as that of the interaction of RafRBD with T35S protein in the absence of any compound as shown in the Figure 3.4.c & e. The kinetic of switching rate from ff state to n state of the T35S protein in the presence of the RafRBD and also in the presence of Zinc cyclen and Lupeol are close to the published rate, which is around 5 sec -1 (Kötting, C., et al., 2007). From the results, it could be implied that similarities are observed between the T35S and Y32W in the presence of RafRBD and Zinc cyclen is usually associated with the increase in the intensities of the γ- phosphates, or conversely n state is more favorable for these mutants in the presence of RafRBD and Zinc cyclen GTPase activity of Ras protein in the presence of the Zinc Cyclen From the Figure 3.5, results indicate the GTPase activity of the Ras protein is not changed significantly in the presence of the Zinc cyclen as predicited by NMR (Spoerner, M., et al., 2005). Absorbance Difference ( ) cm log 10 time (Sec) Figure 3.5 GTPase activity of Ras protein in the presence of the Zinc cyclen. The plot is the normalized values obtained from the absorbance differences between the α phosphate of the GDP (1236 cm -1 ) and α phosphate of the GTP (1262 cm -1 ) vs time. Rate is calculated by first order equation. Control Ras (Black), Ras and Zinc cyclen (Blue, 1X) and Zinc Cyclen (Red, 5X). Rate of the GTPase actrivity is 0.18 min -1, and with zinc cyclen rate is only changed by 2% of the value of the intrinsic rate measured at 303 K.

78 66 The GTPase rates were calculated by plotting the absorbance difference between the intensities of the α GDP and α GTP with respect to time (Kötting, C., et al., 2004). The results from Section and indicate that in the presence of the Zinc cyclen, Ras protein with bound GTP does not undergo any major changes as described by Spoerner, M., et al, In their study (Spoerner, M., et al, 2005), they observed the changes in the conformation of the Ras protein with bound GppNHp nucleotide in the presence of Zinc cyclen, and concluded that the stabilization of a new conformation state was possible with zinc cyclen, which was labeled as State 3 (Figure 1.12). This newly observed State 3 was never seen before from their previous studies on the same nucleotide (GppNHp) studied by 31 P-NMR, and results from Zinc cyclen experiments were proposed into existence of a new conformation state of Ras*GppNHp protein, which has least affinity for the effector proteins and speculated a possibility of improved GTPase activity due to presence of positive charge from Zinc cyclen close to phosphate binding domain. The results from the present study on Ras*GTP by trftir method have shown that such a conformation modulation was not observed in contrast to the NMR studies on Ras*GppNHp. 3.3 Interaction of Ras protein with miscellaneous compounds Interaction of 1,2,3-Hexanetriol with Ras*GTP At concentrations below five times the concentration of 5 mm Ras protein in 10mM Mg 2+, HEPES, at ph 7.5, the compound 1,2,3-Hexanetriol with Ras protein showed well defined kinetics, while above ten times the concentration of 5 mm Ras protein in 10mM Mg 2+ buffer, the protein instantly precipitated. The resulting photolysis difference spectra are shown in the Figure 3.6.a, representing the rate of k 0 + k 1 transition of RasNPEGTP. At higher concentrations, the characteristic peak at 1689 cm -1 is totally absent (Blue), while at low concentration (5X, Red), the characteristic peaks at 1689 cm -1, 1217 cm -1, and 1144 cm -1 are observed similar to that of wild type. The spectra of the 5X and 10X Hexanetriol compounds are also compared with free GTP, which is shown in green (Figure 3.6.a). To probe the role of the 1,2,3-hexanetriol on the dissociation of the nucleotide from the protein, florescence measurements were done with Ras*MantGDP (0.1µM) in the presence of the 200 µm of the 1,2,3-Hexanetriol in HEPES (2 mm), Mg 2+ (5mM) and 100 µm GDP.

79 67 Figure 3.6.a Interaction of hexantriol with Ras*GTP: Photolysis difference spectra of Ras*NPEcgGTP protein in the presence of 1,2,3-hexanetriol (5X-Red) & (10X-Blue) and NPEcaged GTP without Ras protein (Green), measurements were carried in HEPES, ph 7.5, 258 K representing k 0 + k 1. In the presence of the magnesium, 1,2,3-Hexantriol interaction with Ras protein over a period of 10 minutes didn t result in any decrease in the flourescence (Figure 3.6.b, Blue) indicating, that the dissociation of the bound nucleotide from the protein was not achieved in the presence of Mg 2+. In the absence of the Mg 2+, there was decrease in the fluorescence in the presence of the compound 1,2,3-Hexanetriol and Ras protein. The observed result with FTIR was basically a Mg 2+ dependent chelating phenomena by 1,2,3-Hexanetriol. While in the absence of the Mg 2+ (Magnesium free buffer), 1,2,3-Hexanetriol showed the typical nucleotide dissociation behavior similar to EDTA in high concentration (Figure 3.6.b, Black, Red). The present results indicate that nucleotide dissociation could also be achieved not only with EDTA but also with polyhydroxy compounds like 1,2,3-Hexanetriol which destabilize GNB domain by chelating the Mg 2+ cofactor.

80 68 Flourescence Intensity (Fraction) EDTA Hexanetriol (- Mg 2+ ) Hexanetriol (+ Mg 2+ ) Min Figure 3.6.b Ligand-mediated guanine nucleotide exchange of Ras in the presence and absence Mg 2+. Mant-GDP-loaded Ras (0.1 µm) was added, and the decrease in fluorescence emission at 460 nm was recorded as a function of time, with ligands EDTA (15mM), Hexanetriol (200 µm) Switching with Spermidine, Spermine, Putrescene Experiments were done based on the prediction of the existence of hot spots on the protein binding sites (te Heesen, H, 2006) and some the molecules as predicted to interfere with Ras protein were considered. Results indicate in the presence of the equimolar concentration of Mg 2+ and the given compound (Spermidine, Spermine, Putrescene), there are no observable changes associated with switching of the Ras protein from ff state to n state. The kinetics of the switching from ff state to n state (Figure 3.7.a) and also its photolysis difference spectra of the Ras protein in the presence of ten times of spermidine, eight times of Spermine and ten times of Putrescene compared to concentration of the Ras protein are very similar to that of Ras protein without any polyamine compound (Figure 3.7.b). Similar to Zinc cyclen, these polyamines have no role in switching delay or ability in the destabilizing of the switch regions, which might delay either kinetics of switching or the shifting the equlibria to ff state.

81 69 Figure 3.7.a Switching of Ras protein in the presence of Spermidine, Spermine, Putrescene: Rate of switching in the presence of 10X of spermidine (Red), 8X of Spermine (Blue) and 10 X of Putrescene (Green) 6.12 s -1, 6.25 s -1, and 6.14 s -1 respectively, control is shown in black with rate 6.4 s -1. The plot is the generation of the γ phosphate vibration at 1144 cm -1 of the Ras GTP. Figure 3.7.b Interaction of Ras protein with Spermidine, Spermine, Putrescene: Photolysis difference spectra of Ras protein in the presence of 10X of spermidine (Red), 8X of Spermine (Blue) and 10 X of Putrescene (Green), with equimolar Mg 2+, control wild type is shown in black, measurements were carried in HEPES, ph7.5, 258 K, with phpgtp, scaled between cm -1 representing k 0 + k 1.

82 Switching by Acetonitrile (AcCN) and Dimethylsulphoxide (DMS) Multisolvent crystal structures of Ras revealed that stability of the switches could be improved by employing a partial hydrophobic organic solvents (Mattos, C., et al., 2003). Here, experiments with Acetonitrile (AcCN, 1%) and DMS (dimethylsulfoxide, 4%) in the presence of the 12% Ethylene Glycol were conducted to probe the role of the organic solvents in the switching of the Ras protein from ff state to n state. The interaction of the acetonitrile with Ras protein was not favorable at higher percentages, while DMS was tolerated upto 7%. The results of the interaction of the Acetonitrile (1%) and DMS (4%) are shown in the Figure 3.8. The results were compared with respect to wild type Ras protein under similar conditions without any application of either of the Acetonitrile and DMS. Results indicate in the presence of the Acetonitrile (1%) or DMS (4%), the switching kinetics from ff state to n state are perturbed into third order kinetics transistion compared to wild type which undergoes a first order change from ff state to n state (Figure 3.8). In the presence of the acetonitrile (1%) or DMS (4%), the rate of the switching from ff state to n state which is mainly represented in k 2 is delayed by five times compared to control Ras protein without any organic solvent. Rate k 1 and k 3 are difficult to interpret in terms of the switch movement as there is no information regarding the interaction of the Ras protein with organic solvent during ff to n conformation changes. The amplitude spectra of the transition from the ff state to n state are shown in the Appendix 8.6, which correspond to the first two rates. The third slow rate shows a baseline drift associated with change in the water content. From the results, under study with organic solvents like Acetonitrile (1%) and DMS (4%), the kinetic modulation of the switch movement is observed. The surface area of these organic molecules is usually very minute indicating the small molecule which can interfere with the space available between the switch regions is very minute and surface area of the small molecules required to accomplish such modulation should not be larger than the surfaces of the Acetonitrile or DMS. From the MSCS studies (Mattos, C., et al., 2003), most of the organic molecules were oriented close to Switch II regions or other domains which are invariant during surface change by switching mechanism which involve movement of the Switch I and Switch II regions of the Ras protein.

83 71 Figure 3.8 Generation of n state in the presence of Acetonitrile and DMS, the rates are k 1 = 6.6 s -1 and 19.3 s -1, k 2 = 1 s -1 and 0.89 s -1, k 3 = 0.06 s -1 and 0.03 s -1 respectively for AcCN (1%) and DMS (4%). The plot is the generation of the γ phosphate vibration at 1144 cm -1 of the Ras GTP in the presence of acetonitrile (Blue), DMS (Red), Control (Green) using phpgtp with HEPES, ph 7.5, at 258 K. There are no binding pockets on the Ras binding domain of the Switch I region, which are involved in the recognition of the effectors like RafRBD, RalGDS, and also from MSCS studies, there were no organic molecules bound to this interface domain of the Switch I region. During switching mechanism, most of the residues from the Switch I region undergo a transition from open conformation of the switch loop to a closed conformation, also there is a considerable change in the hydrophobicity of the Switch I region, namely Threonine 35, Tyrosine 32 and Isoleucine 36 (PDB: 1QRA, 1PLJ). During the transistion of switching of the Ras protein, the residue Ile 36, whose side chain undergo a complete flipping during switching from ff to n

84 72 state, while Thr 35 and Tyr 32 show the movement of side chains from solvent exposed orientation in the ff state to solvent inaccessable orientation in the n state. The possible delay of the switching kinetics of the Ras protein in the presence DMS or acetonitrile may be due to interaction between the hydrophobic moieties of the protein and organic solvents during the closing of the switch loop regions. 3.5 GTP complex formation with metal macrocycles The following zinc complexes were studied in this section H H H 2 N NH 2 N N N N N N N N H L1 H H 2 N NH 2 L2 N N H NH C H 2 N Fab 7 N N H HN NH Complex formation between GTP and Copper Cyclam Figure 3.9 shows the photolysis difference spectra of the interaction and complex formation of the copper cyclam with free GTP. In the presence of copper cyclam the coupled α,β,γ-phosphate vibrations at 1124 cm -1 totally lost intensity, but, a new peak is observed at 1090 cm -1. The

85 73 negative bands at 1273 cm -1 are downshifted or broadened in the presence of copper cyclam (not labeled in the Figure 3.9) and the characteristic negative peak at 1525 cm -1 of NPEcgGTP is not changed upon interaction with copper cyclam. The results indicate that there is an influence of copper cyclam on GTP orientation on complexation with copper cyclam and shows a perturbation of Mg 2+ coordination, probably destabilizing it. Figure 3.9 Averaged spectra between scans from 400 msec to 10 sec of the total collected 180 scans. Averaged photolysis difference spectra of 3mM NPE caged-gtp (Black) and in complex with 30 mm of Copper cyclam (Red), all spectra were recorded at 258 K. HEPES 10mM, DTT 50mM, MgCl 2 10mM, ph Complex formation between free GTP, Copper Cyclam, Zinc derivative (L1) and Zinc complex of Fab7 As shown in the Figure 3.10, interaction of the copper cyclam, zinc complexes of L1 and Fab7 has shown influence on the spectral properties of the free GTP: Mg 2+. In all cases there is a slight decrease in the intensity of the coupled α,β,γ-phosphate vibrations. When compared to

86 74 interaction of high concentrations of copper cyclam with GTP, in the presence of low concentrations of zinc complexes of L1 & Fab7, only minor changes are observed with L1 and Fab7 compounds. Data imply a concentration dependent behavior of the GTP for the metal phosphate chelate formation. Major changes are only associated with 10X concentrations of the metal complexes, while at lower concentrations there are not many differecenes between free GTP and complexed GTP with metal macrocylces (Figure 3.9 & Figure 3.10). Figure 3.10 Averaged spectra between scans from 1 sec to 20 sec of the total collected 180 scans. Averaged photolysis difference spectra of 3mM NPE caged-gtp (Black) and in complex with 12mM of Copper cyclam (Cyan), in Zinc complex with 12mM of L1 (Blue), in Zinc complex with 12mM of Fab7 (Red). All spectra were recorded at 258 K. HEPES 10mM, DTT 50mM, MgCl 2 10mM, ph Free GTP hydrolysis at 353K in the presence of the compounds (L1, L2, and Fab7) Figure 3.11 shows the influence of the metal complexes on Free GTP and Mg 2+ hydrolysis rates. The ratio between GTP and Mg 2+ is 1:1, while the ratio between GTP and metal complex

87 75 compound is 1:10, in MES 100mM, ph 6.0. Metal complex of L1 increased the free GTP hydrolysis rate by 10 times, while metal complex of L2 and Fab7 were close to 20 times when compared to free GTP. GTP / (GTP+ GDP +GMP + G) min -1 L2 and Fab min -1 GTP 0.13 min -1 L log time (min) Figure 3.11 GTP hydrolysis at 353 K with L1 (Blue), L2 (Green), Fab7 (Red), Control GTP (Black), ratio between GTP and Mg 2+ is 1:1, while the ratio between GTP and compound is 1:10, in MES 100mM, ph 6.0. Reaction was monitored by HPLC. There is no conclusive explanation for the rate increase with FTIR spectra, as free GTP vibrations are broadly coupled and influence of the compound on GTP doesn t produce any significant shifts, but only show differences in the intensities of the coupled vibrations of the α-,β-,γ-phosphates. From the Figure 3.11, the difference in the starting concentration for GTP and in the presence of the compounds is due to the presence of the hydrolyzed products of GTP with L1, L2 and Fab7. So the starting points of the L1, L2 and Fab7 with GTP will never starts at 1.0 for total GTP / (GTP + GDP + GMP + G).

88 GTPase activity of Ras and RasG12V in the presence of zinc substituted macrocycles The promise of the accelerating the free GTP hydrolysis was extended to examine, further the GTPase activity of the Ras*GTP and RasG12V*GTP in the presence of the substituted metal macrocycles. Interaction of the metal macrocycles with Ras*GTP and Ras G12V*GTP were studied by monitoring the single turnover hydrolysis at 30 C, which was analyzed by HPLC. Zinc complex of L1 (300 µm) was studied with Ras*GTP (120µM), and Zinc complex of L2 (320 µm) was studied with RasG12V*GTP (180 µm), respectively, in 5mM Mg 2+, Tris, ph 7.8 at 30 C. The combination of the ligand L2 with respect RasG12V was followed according to the previous studies by Ahmadian, M.R., et al., 1999, where an amino group is involved in the acceleration of the oncogenic mutant. The HPLC data was plotted according to percentage of the GTP available with respect to time under study (Figure 3.12) GTP / Total Minutes Figure 3.12 HPLC measurements of the hydrolysis reaction of Ras-proteins (Ras wild type and RasG12V) loaded with GTP. Single turnover hydrolysis of 120 µm wild-type Ras (Black) and in combination with Zinc complex of L1 (300 µm, Red); Ras G12V (180 µm, Blue) and in combination with Zinc complex of L2 (320 µm, Green), in Tris, ph 7.8, 30 C.

89 77 Results indicate in the presence of the Zinc complexes (L1,& L2) Ras*GTP and RasG12V*GTP hydrolysis rates are not significantly improved at 30 C (Figure 3.12). The GTPase hydrolysis rate of wild type was min -1, while in the presence of the Zinc complex of L1, the rate was close to min -1. n the other hand, GTPase activity of RasG12V was indeterminate as it shows a linear decay in the time period under study for about 5 hours, but profile indicate it is not much different from that of the interaction with the Zinc complex of L2, which show that metal complexes have no role in the modulation of the GTPase activity. Comparison of the results from free GTP hydrolysis and Ras bound GTP hydrolysis implies a different mode of the action by metal macrocyles, where protein bound GTP is not accessable to the metal complexes, while in the free state without any GTPase protein, GTP is shown to interact with metal complexes (Figure 3.9 & Figure 3.10). The success of the downregulation of oncogenic Ras protein was described by Ahmadian, M.R., et al., 1999, where acceleration of the protein bound nucleotide hydrolysis was accomplished by supplying a catalytically functional group into the active site of oncogenic Ras proteins to improve their primary biochemical defects. Results from such studies, where the defective GTPase reaction of oncogenic Ras-mutants were rescued by using DABP-GTP as the bound nucleotide instead of GTP have shown faster hydrolysis rate compared to Ras*GTP. The improved effect of DABP-GTP hydrolysis was attributed to an optimal positioning of the catalytic amine of DABP-GTP, replacing the Gln61 and benefiting from the presence of a hydrophobic patch presented by residues in positions 12/13 of the Ras protein to anchor the DABP-moiety. Later studies revealed that DABP-GTPase mechanism is not related to the intrinsic and GAP accelerated GTP hydrolysis by Ras, and does not involve hydrolysis, but rather an aminolysis reaction. The roles of the catalytic glutamine residue in GTP-binding proteins and the aromatic amino group in DABP-GTP are different, and showed that conclusions on the GTPase mechanism cannot be drawn from findings obtained with DABP-GTP or vice versa (Gail, R., et al., 2001). In the present studies, Zinc complexes of L1 and L2 have shown acceleration of the GTP hydrolysis in the free state not bound to the protein at high temperature, but they have no effect when the nucleotide is bound to the GTPase protein. Results represent a complex phenomena how GTPase proteins orient the functional groups to accelerate the hydrolysis of the protein bound nucleotide, for instance as in the case DABP-GTP, aminolysis was the possibility for

90 78 observed acceleration, but with Zinc complex of L2, NH 2 substituted functionalities were unable to enhance the GTPase activity similar to DABP-GTP amino group. 3.7 Ab initio Calculations on the triphosphate models ptimizations, ESP and energies of the models The optimized structures of the model compounds of triphosphates in the gas phase (T1, T2, T3) with corresponding energies in hartrees (A.U) are shown in the Figure The distances between the equivalent α-phosphate to β Phosphate and β Phosphate to γ Phosphate are denoted by C1 and C2 respectively. From the figure, it can be noted that triphosphate model of T1 is lower in energy compared to T2 and T3. The distances between bridged oxygen from both α-phosphate and β Phosphate (C1) are very similar for all the models (T1, T2, T3), while the distances between the bridged atoms consisting, NH, CH 2 (C2) varied for all the models. The T1 with oxygen as bridging atom between β Phosphate and γ Phosphate has the shortest distance between the phosphate centers compared to T2 and T3. The shorter bond length of all P _ bonds of T1 implies a stronger bond, which is reflected in terms of its lowest energy compared to T2 and T3 (Figure 3.13). The electrostatic potential (ESP) charges of the T1, T2, T3 implies the nature of the charge distribution between the phosphate groups (Appendix 8.7). From the Appendix 8.7, the major differences are present in the T3, where α-phosphate and β phosphate show equivalent charge, while T1 and T2 are similar in ESPs, with γ Phosphate of the T2 showing more positive nature compared to the γ Phosphate of T Frequencies analysis The raw frequency values of phosphates obtained from this study of the model structures T1, T2, T3 at B3LYP/6-31++G** level are presented in this section. Although corrections are needed to obtain accurate frequencies (Scott, A.P, et al., 1996), relative comparison of the raw values can give useful information about the delocalization effect of the bridging atom, NH, CH 2 and also dynamic behavior of the differences between different models (T1, T2, & T3). There are no imaginary frequencies for T1, T2, and T3 which have 48, 51 and 54 normal modes in total respectively. Plots of calculated IR intensities vs. wave numbers for T1, T2 and T3 are presented in the Appendix 8.8. The high intensity modes and their corresponding frequencies (only for

91 79 C1 C2 T1 P P A.U H P T2 P P H N H P A.U T3 P P H H C H P A.U Figure 3.13 ptimized structures and energies of T1, T2 and T3. T1, T2, & T3 represent GTP, GppNHp, and GppCH 2 p analogues respectively. Calculated energies are shown in hartrees (A.U), and calculated bond distances are indicated in blue color. C1 represent the distance between α-, β- phosphates, and C2 represent the distance between β-, γ- phosphates respectively, charges on phosphates are omitted for clarity.

92 80 phosphate groups) are tabulated in the Table 1 respectively for T1, T2, and T3. From the Table 1, importance was given to phosphate vibrations and bridging vibrations between the phosphates, rather than H bonds stretching modes or other functional groups like CH 3. Comparing T1, T2, & T3, it is interesting to note that T1 has the strongest band which is due to coupled vibration of the γ-phosphate to other phosphates without movements of the brdging atom namely oxygen, which was observed at 827 cm -1. While, T2 & T3 shows the bridge stretching and wagging motion of NH and CH 2 as the intense peaks at 863 cm -1 and 881 cm -1 respectively. Table 1 Energies of the normal modes in cm -1 and intensities in the brackets of the calculated T1, T2, and T3 models (calculated IR is shown in the appendix 8.8). Important normal modes of phosphates models are tabulated. Mode of Vibration α-phosphate β-phosphate γ-phosphate bridge stretch T1 T2 T3 1270, 1216(41) 1220(43) c 1230 b, 1219 b, 1171(38) 1198(41) c, 1150, 827(30) e 994(33) d,1120(37) d 1024(34) d, 914(31), 863(32) d, 805(31) 881(31) d,735(28) (Note, b = coupled vibration between γ-phosphate and β-phosphate without movement of the bridged atom, c = coupled vibration α-phosphate and β-phosphate, d = coupled vibration between γ-phosphate and β-phosphate with wagging or out of plane stretch movement of the bridged atom, e = coupled vibration between all phosphates without movement of the bridged atom). Results from the calculation (Table 1) of the frequencies shows that model T2 and T3 have different modes of movement of the bridging atom namely, NH and CH 2 where coupled vibration between γ-phosphate and β-phosphate show wagging or out of plane stretch movement of the bridged atom, while such movement was not observed from the calculation on the model phosphate of T1, which is a true representation of the natural nucleotide.

93 trftir studies on non-hydrolyzable GTP analogs and GDP bound to Ras protein Photolysis difference spectrum and kinetics of Ras*GppCH 2 p Results from the photolysis of the available nonhydrolyzable caged GppCH 2 p (Jena Bioscience) indicate the behavior of the GppCH 2 p bound to Ras protein is similar in the amide regions as that of GTP bound to the Ras protein (Figure 3.14.a). The phosphate regions of the Ras*GppCH 2 p are quite different from that of Ras*GTP peaks corresponding to γ phosphate and β-phosphate vibrations. The assignment of the phosphate regions of the Ras*GppCH 2 p was not possible due to nonavailability of the labeled phosphates of the GppCH 2 p. The similarity in the amide regions of the Ras*GppCH 2 p compared to Ras*GTP and the strong deviation from the amide region of the T35A ff conformation could be a possibility that the nonhydrolyzable bound form of the Ras*GppCH 2 p protein is in the n conformation state. Figure 3.14.a Comparison of photolysis difference spectra of nonhydrolyzable and hydrolyzable nucleotide, Ras*GTP (hydrolyzable analog, Black), Ras*GppCH 2 p (Red), Ras T35A*GTP (Blue). Characteristic Ras*GTP peaks are labeled; measurements were carried at 258 K in HEPES, ph 7.5, scaled at 1345 cm -1.

94 82 Figure 3.14.b Switch movement in Ras bound nonhydrolyzable nucleotide: Comparsion of the highest phosphate peaks to the Threonine-35 switch I (1689) backbone movement of the Ras*GTP (3.2 s -1 ) and Ras*GppCH 2 p (5.2 s -1 ). Despite the problem, that only NPE caged GppCH 2 p was available, a kinetic analysis was attempted. Kinetics of the significant peak of the backbone vibration from the switch I movement of the Threonine 35 at 1689 cm -1 of the Ras*GTP are plotted with respect to Ras*GppCH 2 p (Figure 3.14.b) and compared with repective peaks in the phosphate regions with high intensity, namely 1144 cm -1 for Ras*GTP and 1085 cm -1 for Ras*GppCH 2 p. Results indicate the behavior of the switch I movement of the Thr-35 backbone vibration from the Ras*GppCH 2 p at 1689 cm -1 doesn t posses any kinetic behavior similar to the hydrolyzable nucleotide like Ras*GTP s characteristic peak at 1689 cm -1, and also the peaks with high intensity in the nonhydrolzable nucleotide of Ras*GppCH 2 p at 1085 cm -1 has no well defined behavior (Figure 3.14.b). Results from the Figure 3.14.b, indicate that in comparison to Ras*GTP, the nonhydrolyzable nucleotides like GppCH 2 p do not exhibit well defined kinetic

95 83 behavior between switch I backbone movement during switching from ff state to n state and corresponding reorientation of the phosphate moieties. Also from the Figure 3.14.a, one of the identical peak at 1614 cm -1 from both of the Ras* *GTP and Ras* *GppCH 2 p was selected and furtherr kinetically analyzed as shown in the Figure c. Results indicate that the amide region corresponding to the switch movement at 1614 cm -1, was identical in the either case, which means the part of the switch region corresponding to peak at 1614 cm -1, undergoes an equivalent structural transition from ff state to n state on interaction with either GTP or GppCH 2 p (Figure 3.14.c). Apart from the comparison of the kinetic behavior of the switch movement of Ras*GTP and Ras*GppCH 2 p, amplitube spectra corresponding to the conformational changes involved during the closing of the switch regions from the ff state to the n state were compared as shown in the Figure 3.14.d. Figure 3.14.c The plot is the generation of the n state vibration at 1614 cm -1 of the Ras* *GTP (Black), & Ras*GppCH 2 p (Red). Measurements were carried at 258 K in HEPES, ph 7.5, NPE caged was used.

96 84 From thee Figure d, results indicate thee rate k1 corrresponding to transitionn from the ff state to thhe n statte, show som me notable siimilarities inn the amide regions, r withh an exception of the Threoonine-35 bacckbone moveement vibrattion at 1689 cm-1. In the am mide regionss, the positivve peaks corrresponding to the Ras**GTP and Ras*GppCH R 2p at 1611 cm m-1, 1564 cm-1, and cm-1 were identical in the positionn of the amidde bands, which w representt the n sttate generatiion, though the t observedd intensities are variablee (Figure d). While coomparison of o the peak at a 1689 cm-1 from both nucleotidess are shown with an oppposite intensity for Ras*G GTP and Ras*GppCH R ng a slightt different behavior off the 2p, indicatin Threoninne-35 backbo one movemeent, during the switchinng from fff state to n state of o the Ras*GTP P and Ras*G GppCH2p resspectively (F Figure 3.14.d d). 3 Confo ormation chaanges involved during thhe switching of the Ras*GTP (Blackk) and Figure 3.14.d Ras*GpppCH2p (Red)) from the ff state to the n staate representting the rate k1.

97 Interaction of Ras*GppCH 2 p with Zinc cyclen As shown in the Figure 3.15 a, b, interaction of the GTP analog GppCH 2 p with Ras protein is studied under different conditions. In the Figure 3.15.a, Ras*GppCH 2 p is studied under different temperatures and it is also compared with interaction of Zinc cyclen with Ras*GppCH 2 p. From the results, it could be seen, that there are changes associated with temperature on the phosphate regions around 1170 to 1050 cm -1. Change in the temperature of the measurement between 258 K and 273 K has effect on the intensities of the phosphate regions of the Ras*GppCH 2 p. As described earlier in the result from the Section 3.8.1, there is no well defined kinetic bahavior of the switch movement corresponding to the positive peaks at 1689 cm -1 and 1085 cm -1 from Ras*GppCH 2 p, though the spectra of the Ras*GppCH 2 p in the amide regions are similar to that of the Ras*GTP. Figure 3.15.a Photolysis difference spectra of the Ras*GppCH 2 p (Red, 273 K), Ras*GppCH 2 p with Zinc Cyclen (Green), Ras*GppCH 2 p (Blue, 258 K).

98 86 For Ras*GTP, which show minor or no changes in the phosphate regions with change in the temperature, while for the Ras*GppCH 2 p, intensities are quite different with change in the temperature especially at the position 1085 cm -1. The kinetic analysis of the change in intensities revealed the behavior of the amide switch regions and phosphate moieties are having different mode of first order transistion rate. In the case of the 1689 cm -1 and 1614 cm -1, the behavior is insignificant with change in the temperature, while 1085 cm -1 and 1527 cm -1 (not assigned yet) show significant changes in the kinetics. The possible phosphate moiety peak at 1085 cm -1 shows an increase in the intensity with respect to increase in the temperature, while 1527 cm -1 peak shows variable kinetic rate with temperature (Figure 3.15.b), which indicate a possible equilibrium shift in the Ras conformation states. Figure 3.15.b Kinetic analysis of the nonhydrolyzable nucleotide at different temperatures, the major peaks which were changing in intensities during transistion was plotted at different temperature.

99 87 In the presence of the zinc cyclen, the amide regions are perturbed to a large extent, while in the phosphate region, the changes are observed in the intensities.the characteristic amide regions are perturbed in the presence of the zinc cyclen, which indicate a process where the closed switch I region is opened without any coordination of the threonine to Mg 2+ and γ-phosphate of the nucleotide. The results of interaction of zinc cyclen, with the Ras*GppCH 2 p, are shown in the Figure 3.15.a the spectra show the influence the phosphate orientations in the presence of the metal macrocycles which can act as phosphate chelates. The changes indicate a fluxional nature of the switch regions of the protein bound to nonhydrolyzable nucleotides like GppNHp and GppCH 2 p which could be perturbed with phosphate chelating metal macrocycles and also intensities of the phosphate regions are changing with change in the temperature Interaction of Copper Cyclam with Ras*GDP Figure 3.16 shows the interaction of Ras*GDP with copper cyclam. In the presence of the copper cyclam (4X of Copper cyclam, HEPES 100mM, 5mM MgCl 2, 10mM DTT, ph 7.5) Figure 3.16 Averaged spectra of interaction of copper cyclam with Ras*GDP. Spectra were averaged between scans from the total collected 180 scans in the time scale of 400 msec to 40 sec carried at 258 K. Averaged photolysis difference spectra of Ras*cgGDP (Black) and Ras*cgGDP with Copper cyclam (Red).

100 88 amide regions were totally disturbed, while average between spectra of the total 180 collected spectra in the time scale of 400 msec to 40 sec revealed changes which are hard to interpret as there is no kinetic rate phenomena for Ras*GDP phosphate regions. The possible explanation of the result is the interaction of the metal cyclam with phosphate moiety, as GDP bound state of Ras is known to exist in couple of open ff state conformations with variable switch regions. While Ras*GTP has not shown this sort of phenomena in the presence of Zinc cyclen indicating that switch regions in the Ras*GTP state exist in only one n state in constrat to NMR studies on Ras*GppNHp (Spoerner, M., et al, 2005a). 3.9 Metal NF1 peptide bioconjugates Synthesis of the Ruthenium NF1 peptide bioconjugate The strategy involved in the synthesis of the Ruthenium NF1 peptide biocojugate is depicted in the Scheme 2. Synthesis involves a monosubstituted cyclam (L3), NF1 peptide fragment and followed by coupling of L3 and NF1 peptide fragment resulting NF1 peptide bioconjugate, which further incorporates a metal in the formation of metal NF1 peptide bioconjugate. Monosubstituted cyclam (L3) The steps in the synthesis involves the generation of the linker macrocycle ligand L3, which is formed by reaction of cyclam with p-bromomethyl benzoic acid. The reaction yield for this one step conversion into monosubstituted cyclam is around 70%, and corresponding mass analysis with electrospray ionization in positive mode [M+H] + showed the peak at confirmed the identity of the ligand L3 as shown in the following Figure 3.17.a. NF1 peptide fragment Gly-Gly-Gly-Thr-Leu-Phe-Arg 789 -Ala-Thr-Thr-Leu- Ser-Arg The NF1 peptide product after ESI + analysis showed the presence of the required peptide with the sequence Gly-Gly-Gly-Thr-Leu-Phe-Arg 789 -Ala-Thr-Thr-Leu-Ser-Arg, the mass corresponding to the required sequence is a doubly charged entity with peak at There are

101 89 also entities of the peptides with longer and shorter sequences than required NF1 peptide fragment. Most notably the sequences with shorter peptides have truncated glycines at N- terminus are observed. The resulting truncated peptides have the sequences corresponding to Gly-Thr-Leu-Phe-Arg 789 -Ala-Thr-Thr-Leu-Ser-Arg and Gly-Gly-Thr-Leu-Phe-Arg 789 -Ala- Thr-Thr-Leu-Ser-Arg respectively, with ESI + doubly charged masses corresponding to and (Figure 3.17.b) Intensity Intensity Mass Figure 3.17.a) ESI of the Ligand L3 Mass.b) ESI of the NF1 peptide fragments NF1 peptide bioconjugate The purity of the resulting NF1 peptide bioconjugate was analyzed by HPLC (Figure 3.18.A), the results indicate that NF1 peptide bioconjugate is not the sole product. It consists of a mixture of peptide conjugates. The peptide conjugates were collected according to retention time into three fractions from 7 to 8 minutes, 8.5 to 13 minutes and from 13 to 16 minutes respectively (Figure 3.18.A). Each fraction was analyzed by ESI +. The fraction collected between 8.5 min to 13 minutes has the required peptide conjugates, while the last fraction conjugates seems to have high molecular mass peptide conjugates. Mass analyses of the required fraction confirmed the identities of the NF1 peptide bioconjugate with doubly charged masses at respectively, there are also masses corresponding to the truncated glycines (794.40) (Figure 3.19.a, Appendix 8.9).

102 Figure 3.18 A. HPLC analysis of the synthesized NF1 peptide conjugates, B. HPLC analysis of the synthesized Ruthenium NF1 peptide bioconjugates mixture, C. HPLC analysis of the purified fraction from Ruthenium NF1 peptide bioconjugates without truncation of glycines, D. HPLC analysis of the truncated Ruthenium NF1 peptide bioconjugates. 90

103 Intensity Mass Figure 3.19.a Electrospray analyses of the doubly charged NF1 peptide bioconjugates purified from the Figure 3.18.A, the spectrum represents the fraction collected between the intervals 8.5 to 13 minutes. Ruthenium NF1 Peptide bioconjugate During purification, Ruthenium NF1 peptide bioconjugate was collected into two fractions between the retention times ranging from minutes and minutes (Figure 3.18.B). Coordination of the metal to peptide bioconjugates results in the increase of the retention time of the product which is indicated in the Figure 3.18.B. The first fraction is the required peptide conjugate without any truncated glycines with retention time at 13.1 min (Figure 3.18.C), while the second fraction (Figure 3.18.D) has truncated glycines at 16.2 min. Electrospray analysis of the fractions after purification revealed the identities of the peptides as ruthenium coordinated bioconjugates with typical signature of ruthenium isotopic distribution with masses , , and (Figure 3.19.b, Appendix 8.9). The resulting Ruthenium NF1 bioconjugates synthesized and confirmed by ESI + are shown in the Figure 3.19.c. The purity of the final metal NF1 peptide bioconjugate without truncation is around 80 %, and remaining portion is found

104 92 with uncomplexed NF1 peptide conjugate. Importance was given to both of the fractions which consist three of the Ruthenium NF1 bioconjugates (Figure 3.19.c- B, C, D), as any of the fractions might have any GTPase activity improvement m/z Figure 3.19.b Electrospray analyses of the doubly charged Ruthenium NF1 peptide bioconjugates purified at 13.1 min from the Figure 3.18.C, the spectra represents the ruthenium bioconjugates are shown in the Figure 3.19.b trftir measurements of GTPase activity with NF1 peptide, NF1 peptide conjugate and Ruthenium NF1 peptide bioconjugate Due to insolubility of the NF1 peptide and NF1 peptide conjugate, only experiments with 0.2 X concentrated peptide mixtures with respect to Ras protein were performed. Results from interaction with 0.2X mixture in water constituting peptide and peptide conjugate indicate that

105 93 NH X M N Gly Gly M = Ru, X= H,Cl Gly Thr Leu A Phe Arg Ala Thr Thr Leu NH X HN Ser Arg NH NH H Ru H N HN Na+ Gly Gly Gly Molecular Weight: Thr Leu B Phe Arg Ala Thr Thr Leu Ser Arg NH H N Gly Gly Thr Leu Phe Arg Ala Thr NH Ru H HN Molecular Weight: C Leu Thr Arg Ser NH H N Gly Thr Leu Phe Arg Ala Thr NH Ru H HN Molecular Weight: D Leu Thr Ser. Arg Figure 3.19.c Metal bioconjugates consisting of NF1 catalytic residue Arg 789, A = general chemical structure of the metal bioconjugate, B,C,D = Metal NF1 peptide bioconjugates observed after the solid phase synthesis and mass spectra analysis

106 94 Figure 3.20.a Photolysis difference spectra of Ras wt NPE cggtp with 1% NF1 (Black), and in the presence of mixture of 0.2X NF1 peptide conjugate compared to Ras protein and conjugate mixture (Red) consituting critical Arginine 789 compared to Ras protein. Measurement was performed at 283 K, HEPES, ph 7.5, scaled at 1345 cm -1. there is no significant improvement on the nature of GTPase activity at 283 K. A photolysis difference spectrum of the peptide mixture (Figure 3.18.a, retention time min) is similar to that of the wild type spectra in the phosphate regions (Figure 3.20.a). Kinetics associated with GTPase activity in the presence of the peptide conjugates was plotted (Figure 3.20.b). Compared to 1% NF1 assisted GTPase activity, 0.2X mixture of NF1 peptide and NF1 peptide conjugates has no GTPase activity at 283 K, while, GTPase activity of wild type with 2X of ruthenium cyclam complex of L3 at 303 K is not affected (not shown), which shows that ruthenium cyclam complex has no interference with the GTPase activity of the wildtype. It can be assumed, that it is not valid to compare the rates or results at two different temperatures, as at low temperatures Ras wild type intrinsic GTPase activity rate is too slow, and interaction with 0.2X peptide and peptide conjugate mixture is not optimal similar to the pure NF1 protein.

107 Absorbance Difference k = 0.04 sec log 10 time (Sec) Figure 3.20 b Kinetic analysis of the GTPase activity of the 1% NF1 with wild type and NF1 peptide fragment with wild type at 283 K (Black), GTPase activity in the presence of 0.2X mixture of NF1 peptides and NF1 peptide conjugates (Red). Plot is the difference between the intensities of the α-phosphate vibrations of GDP (1236 cm -1 ) and α-phosphate vibrations of GTP (1262 cm -1 ). Kinetics are fitted to simple first order exponential decay. Results from the Figure 3.20.b, indicate that mere presence of NF1 catalytic sequence, Gly-Gly- Gly-Thr-Leu-Phe-Arg 789 -Ala-Thr-Thr-Leu-Ser-Arg containing the critical arginine residue 789 is not enough to perform the acceleration of the GTPase activity. In the intrinsic conditions, the GTPase activity of the Ras wild type in the presence of two times of Ruthenium NF1 peptide bioconjugates (Figure 3.19.c B,C,D) with respect to Ras protein at 303 K has a net times of GTPase activity inhibition compared to wild type under similar conditions (Figure 3.20.c). The photolysis difference spectra of Ruthenium NF1 peptide bioconjugates at 303 K is strikingly similar to the Ras wild type, only with minor changes in the amide regions, and it is difficult to explain why the intrinsic GTPase activity is inhibited in the presence of the Ruthenium NF1 Peptide bioconjugate with only small changes in the amide regions (Figure 3.20.d), thus there is interaction of the NF1 peptide conjugate mixture with Ras protein.

108 96 Absorbance Difference, ( ) cm log 10 time (sec) min min -1 Figure 3.20.c Kinetic analysis of the GTPase activity of wild type at 303 K (Black), GTPase activity in the presence of 2X Ruthenium NF1 peptide bioconjugates (Red). Plot is the difference between the intensities of the α-phosphate vibrations of GDP and α-phosphate vibrations of GTP. Kinetics is fitted to simple first order exponential decay. Figure 3.20.d Photolysis difference spectra Ras wt NPE cggtp (Black) and in the presence of 2X of Ruthenium NF1 peptide bioconjugate (Red). Measurement was performed at 303 K, in HEPES, ph 7.5.

109 97 Studies with Ruthenium NF1 bioconjugate (2X) were also conducted at 303 K on Q61A, a typical oncogenic protein which lacks intrinsic and GAP stimulation. In the presence Ruthenium NF1 peptide bioconjugate, the intrinsic GTPase activity of the Q61A is not improved and there was no GTPase activity under measurement time. The attempt to measure GTP hydrolysis by RasQ61A shows a baseline drift with no characteristic phosphate bands, indicating that there is no role of the metal bioconjugate in the improvement of GTPase activity on the Q61A mutant as this mutant didn t showed any hydrolysis during the measurement time (24 hr) at 303 K (Appendix 8.10) GAP assisted GTPase activity of Ras in the presence of 1% NF1 and Ruthenium NF1 peptide bioconjugate Experiments similar to the intrinsic conditions were repeated again with Ruthenium NF1 peptide bioconjugates and Ras protein in the presence of the 1% NF1 at 283 K. In the presence of the 1% NF1, Ruthenium NF1 peptide bioconjugate has net inhibition of GTPase activity by 10 times (Figure 3.20.e). The changes in the photolysis difference spectra are mainly in the intensities of the γ-phosphate vibrations around 1156 cm -1 and a small blue shift in the β phosphate vibrations (Figure 3.20.f). The minute changes observed in the phosphate regions around 1217 to 1155 cm -1 and amide regions at 1614 cm -1 may be due to competition between the metal peptide bioconjugate and NF1 protein, though this sort of assertion is not conclusive as the mode of binding of the Ruthenium NF1 peptide bioconjugate to Ras protein is not known at present and it is not in the scope of this thesis to crystallize this metal peptide bioconjugate with the Ras protein. Compared to minute changes observed in the phosphate regions of the protein bound GTP, the hydrolysis difference spectra has no significant changes in the phosphate regions of the proteins, while amide regions are perturbed with respect to Ras wild type (Figure 3.20.g). Small changes associated with interaction of Ruthenium NF1 peptide bioconjugate with Ras protein and resulting inhibition of the GTPase activity is hard to interpret as there is no information regarding the influence of such metal peptide bioconjugates on GTPase protein function yet or there is no information on the mode of interaction with Ras protein.

110 k = 0.02 sec k = sec log 10 time ( sec ) Figure 3.20.e Kinetic analysis of the GTPase activity of the 1% NF1 with wild type at 283 K (Black), GTPase activity in the presence of 2X Ruthenium NF1 peptide bioconjugate (Red). Plot is the difference between the intensities of the α-phosphate vibrations of GDP and α-phosphate vibrations of GTP. Kinetics is fitted to simple first order exponential decay with substracted intensities normalized. Figure 3.20.f Photolysis difference spectra Ras wt NPE cggtp in the presence of 1% NF1 (Black) and in the presence of 2X of Ruthenium NF1 peptide bioconjugate (Red). Measurement was performed at 283 K, HEPES, ph 7.5, scaled at 1345 cm -1.

111 99 Figure 3.20.g Hydrolysis difference spectra of Ras wild type with 1% NF1 (Black), Ras wild type in the presence of 2X Ruthenium NF1 peptide bioconjugate (Red). Measurements were done at 283 K in HEPES, ph 7.5 with 1% NF1 protein Study of switching in the presence of Ruthenium NF1 peptide bioconjugate To examine whether there is a possible mode of interaction of Ruthenium NF1 peptide bioconjugate with Ras protein on its binding domain with effectors, the Ruthenium NF1 peptide bioconjugate was studied during the switching kinetics from the ff state to the n state. Experiments were conducted with T35S in the presence of RafRBD, and results indicate in the presence of 2X of Ruthenium NF1 peptide bioconjugate with respect to T35S protein and RafRBD, the final n state is not perturbed indicating this metal bioconjugate probably doesn t interfere with RBD of the Ras protein (Figure 3.20.h).

112 100 Figure 3.20.h Interaction of the effector RafRBD with Ras T35S*GTP in the presence of the 2X of Ruthenium NF1 peptide bioconjugate: photolysis difference spectra of T35S and RafRBD (Black), T35S, RalGDS and 2X of Ruthenium NF1 peptide bioconjugate (Red), in HEPES, ph 7.5, 258 K, phpgtp was used, & scaled at cm -1. Changes are observed in the γ-phosphate intensities and around cm -1 of the amide regions with out any significant band shifts. Compared to the Zinc cyclen interaction with T35S and RafRBD, which has shown a blue shift of 8 cm -1 for the β phosphate vibration, there was no comparative shift observed with Ruthenium NF1 peptide biocojugate. The results indicate that different metal have different effect on the β phosphate vibration, where Zinc cyclen is a classical phosphate chelate, while Ruthenium is known to be have interaction with carboxylates. The rate of switching from ff state to n state is shown in the Figure 3.20.i, which indicate the closing of the switch I loop is not affected in the presence of the Ruthenium NF1 peptide bioconjugate and it is attained with a rate of 4.2 sec -1 in the presence of Ruthenium NF1 peptide bioconjugate, while in the absence of the metal peptide bioconjugate, the rate was at 4.7 sec -1.

113 101 Absorbance Difference, 1144 cm log 10 Time (Sec) Figure 3.20.i Kinetics of the generation of the normalized γ phosphate vibration of T35S at 1144 cm -1 in the presence of the effector RafRBD. Ras T35S*phpGTP and RafRBD (control, 4.7 s -1 ), 2X of Ruthenium NF1 peptide bioconjugate (w.r.t to GTPase) in the presence of the RasT35S*phpGTP and RafRBD, rate 4.2 s -1. Results indicate that in the presence of the Zinc cyclen or Ruthenium NF1 peptide biconjugate, the inherent interaction between the Ras protein and its cognate binding effector partners are not affected, which shows metal complexes have least influence on the surface of the Ras binding domain where effectors have interface interaction with switch I region Nitrosation of Ras Protein. Under the presence of the N 2, Ras protein has a tendency to undergo a free radical propagated reaction on the bound nucleotide which is assisted by the Cysteine-118 thyiyl radical and stabilization of the Phenylalanine-28 n-π* interaction with the bound nucleotide (Heo, J., et al., 2004, 2005a, 2005b, 2005, 2006). The redox reaction is a concentration dependent process where high concentration of the N and 2 results in the degradation of the bound nucleotide and also nitrosation of cysteine residue 118 which blocks further reaction of the protein or nucleotide towards nitric oxide and oxygen. Ras protein and S-nitrosylated Ras protein doesn t differ in its

114 102 structural and biochemical properties (Williams, J.G., et al., 2003). Here the task is undertaken to address the role of the nitric oxide and oxygen under basal conditions (low concentrations) where the protein is not precipitated and doesn t undergo any S-nitrosylation of the Cysteine 118 or any other chemical transformations on the bound nucleotide. Further possibilities of anticancer applications are explored in terms of dissociation of the active form of the nucleotide bound Ras protein Photolysis studies of N and 2 release from metal complexes The generation of the nitric oxide and oxygen is carried out by photolysis of the Potassium pentachloronitrosoruthenate(ii) chloride and Titanium peroxo citrate respectively. Figure 3.21, shows the photolysis difference spectrum of generation of the nitric oxide and oxygen from the metal complexes and results in the formation of nitrogen oxides observed in the regions of cm -1. The characteristic peak in the generation of N is at 1880 cm -1, which is a negative peak resulting from the cleavage of the bond between nitric oxide and metal bond from the Ru- N complex, while the labeled N at 1848 cm -1 shows a red shift of 32 cm -1 on nitrogen labeling to 15 N. At present Titanium peroxocitrate is not well characterized in terms of its oxygen release mechanism, as it is very latest compound of stable peroxo complexes and also most of the peroxo complexes are very difficult to elucidate the photolysis mechanism. It is not in the scope of this thesis to characterize the bond positions of the titanium peroxo citrate as this is the PhD project of Markus Rohe (Anorganische Chemie- I, Ruhr Universitat Bochum). For complete photolysis of the Ruthenium Nitrosyl complex around flashes (308 nm / 90 mj energy per flash) were required; while for the titanium peroxocitrate required flashes. In the presence of the oxygen from titanium peroxo citrate, nitric oxide reacts to form complex mixture of nitrogen oxides as represented by the peaks around cm -1. In the presence of labeled nitric oxide ( 15 N), the peaks are shifted by around 50 cm -1 and the resulting peaks are observed at cm -1. The decrease in the intensity of the nitrogen oxides in the unlabeled experiment at cm -1 is due to overlap of the negative peaks resulting from the photolysis of the titanium peroxocitrate (Appendix 8.3.a). Individual photolysis difference spectra s of the metal complexes are shown in the Appendix 8.3.a,b.

115 103 Figure 3.21 Generation of nitrogen oxides from nitric oxide and oxygen. Photolysis difference spectra of the Potassium pentachloronitrosoruthenate (II) chloride and Titanium peroxo citrate, unlabeled nitric oxide (Blue) and labeled nitric oxide (Red) Interaction of N, 15 N and 2 with Ras*GDP To probe the role of nitrogen oxides in the Ras protein signaling, experiments were carried on Ras*GDP in DTT and DTT free conditions. Results are shown in the Figure 3.22.a and 3.22.b, represent the interaction of the nitric oxide and oxygen in the absence and presence of DTT. The characterstic peaks observed in the Figure 3.21 are also observed, indicating the generation of the nitrogen oxides from nitric oxide and oxygen at cm -1. While for the labeled nitric oxide, the nitrogen oxides are downshifted by 50 cm -1 and observed at cm -1. In the absence of the DTT, the spectral differences are seen between the labeled and unlabeled nitric oxide experiments in the regions of cm -1 in the presence of oxygen, indicating possible changes associated with interaction of the nitrogen oxides with Ras bound nucleotide (Figure 3.22.a). In the other case, in the presence of DTT, both of the labeled and unlabeled nitric oxide spectra look very much similar in cm -1 region, indicating the scavenging of the free radicals (Figure 3.22.b). The inability to generate free radicals in the presence of

116 104 Figure 3.22.a Photolysis difference spectra of interaction of nitric oxide and oxygen with Ras*GDP without DTT: measurement was carried at 258 K, in HEPES, ph 7.5. Ras*GDP interaction with unlabeled nitric oxide is shown in Blue, and oxygen, while Ras*GDP in the presence of labeled nitric oxide and oxygen is shown in Red. Figure 3.22.b Photolysis difference spectra of interaction of nitric oxide and oxygen with Ras*GDP in the presence of DTT: measurement was carried at 258 K, in HEPES, ph 7.5, 5mM DTT. Ras*GDP interaction with unlabeled nitric oxide and oxygen (Blue) and Ras*GDP in the presence of labeled nitric oxide and oxygen (Red).

117 105 DTT or ascorbate was previously shown to negate the effect of nitric oxide mediated nucleotide dissociation (Heo, J., et al., 2005a, b). The conclusions from this study show, that in the presence of the N and 2, redox active forms of the GTPases could be perturbed in the absence of any free radical scavengers like DTT Protein bound nucleotide degradation on UV Irradiation Freshly nucleotide exchanged Ras protein (Ras*GppNHp) or without exchange as with Ras*GDP without any DTT was incubated with 10 times of Ruthenium Nitroso complex and 5 times of Titanium peroxo citrate. The reaction was analyzed by HPLC before and after UV light irradiation. After irradiation for minutes, the resulting protein was completely precipitated. Whole reaction content was filtered through amicon concentrator (10 KDa cut off). The filtrate was analysed by HPLC (Figure 3.23). Ras*GDP Ras*GppNHp Absorbance, 254 nm flowthrough after UV reaction Time (min) Figure 3.23 HPLC analyses of the reaction products from nitrosation of the nucleotide with 5% AcCN, Ras*GppNHP (Pink), Ras*GDP (Cyan), reaction flowthrough from the reaction of nitric oxide and oxygen generated by ruthenium and titanium complexes on Ras*GDP (Green).

118 106 The reaction flowthrough was analyzed by electrospray ionization. Results indicate the presence of oxidation and nitration products of the nucleotide GppNHp and GDP respectively (Appendix 8.11). The interaction of metal macrocycles also resulted in the cleavage of the phosphate bonds as discussed in the results from the Section 3.5, interaction of the free nucleotide with copper cyclam and substituted metal macrocycles as shown the cleavage of the phosphate groups. The final products observed on nitration and oxidation of the Ras protein are 5- substitiuted nitro and oxo guanosine derivatives as described in the Appendix 8.11, which are in conformity with the published data (Heo. J., et al., 2005b, 2005) (Appendix 8.11) and also precipitated Ras protein was not analyzed further Intrinsic GTPase activity in the presence of nitrosation environment Under basal conditions which represent the low concentrations of nitric oxide and oxygen, intrinsic GTPase activities were measured in the presence of oxygen and also with nitric oxide and oxygen in the DTT free conditions. Results were compared with that of control experiment without any of the oxygen and nitric oxide under nonprecipitation and also in the presence of DTT under similar conditions of nitric oxide and oxygen. The characteristic peaks of the Ras protein are retained without any band shifts in the presence of the oxygen notably γ phosphate vibration at 1144 cm -1, β-phosphate vibration at 1216 cm -1 and the switch backbone vibration from the threonine-35 at 1689 cm -1 (not shown for clarity), though some changes are observed in the amide II regions (not shown for clarity) (Figure 3.24.a). The motive of discussing the amide regions in the presence of 2, or N and 2 is not of interest as presence of three caged compounds makes it difficult to come to an effective conclusion and also presence of the side reaction between N and 2 makes it further difficult to interpret amide regions. In the presence of oxygen (0.4X), γ phosphate and β-phosphate vibrations are blue shifted by 2 cm -1 with unsual broad shape around 1144 cm -1, due to overlap of the photolysis spectra of oxygen as shown in the Figure 3.24.a in blue color. In the presence of nitric oxide (0.5X) without any oxygen, there are no changes in the phosphate regions with respect to Ras protein as control (Figure 3.24.a, Yellow), Ras protein as a control is shown in the black color. Under DTT free conditions, the effect of nitric oxide (0.5X) and variable concentration of oxygen on the Ras protein was studied with change in the concentration of the oxygen at 0.4X (Figure 3.24.a, Red) and 0.2X (Figure 3.24.a, Green) respectively.

119 107 Figure 3.24.a Photolysis difference spectra of the Ras protein in the presence of nitrosation environment: Ras protein control (Black); Ras protein and oxygen (2X) (Blue); Ras protein, nitric oxide (0.5X) and oxygen (0.4X) (Red); Ras protein, nitric oxide (0.5X) and oxygen (0.2X) (Green); Ras protein and Nitric oxide (2X, Yellow); Ras protein, nitric oxide (0.5X) and oxygen (0.2X) in the presence of DTT (Pink). Measurement was carried at 298 K, in HEPES, ph 7.5, NPEcgGTP was used. Most of the characterisitic peaks of the Ras protein are retained in the presence of basal concentrations of nitrogen oxides without any precipitation of the protein. ne of the notable difference is in the region of the β phosphate vibrations, whose intensity is diminished and also found to be blue shifted to 1221 cm -1 in the presence of the nitric oxide (0.5X) and oxygen (0.4X) (Figure 3.24.a, Red). In the other case with nitric oxide (0.5X) and oxygen (0.2X) (Figure 3.24.a, Green), there was a small red shift of 1 cm -1 at both of the β and γ phosphate vibrations without any precipitation of the Ras protein. In the presence of DTT, the photolysis difference spectra of Ras protein in the presence of nitric oxide (0.5X) and oxygen (0.2X) looks similar to control Ras (Figure 3.24.a, Pink).

120 108 Absorbance Difference, cm Control 2 N DTT, N + 2 N (0.5X) + 2 (0.2X) N (0.5X) + 2 (0.4X) log 10 time (sec) Figure 3.24.b GTPase activity rates in the presence of nitric oxide and oxygen at 298 K: Ras Control (Black, min -1 ); Ras and oxygen (2X) (Blue, min -1 ); Ras & niric oxide (0.5X) & oxygen (0.4X) (Red, Inactive); Ras & nitric oxide (0.5X) & oxygen (0.2X) (Green, min -1 ); Ras & nitric oxide (2X) (Yellow, min -1 ); Ras with DTT & niric oxide (0.5X) & oxygen (0.2X) (Pink, min -1 );. Rates are fitted to first order exponential for absorbance difference of α-gdp and α-gtp vs time. Measurements in HEPES, ph 7.5, 298 K. Results indicate that in the presence of oxygen without any of the nitric oxide; or vice versa, the intrinsic GTPase activity of the Ras protein is not altered at 298 K (Figure 3.24.b, Blue). GTPase activity of the Ras protein in the presence of redox reactive species of oxygen and nitrogen is summarized in the Table 2.

121 109 Table 2. Rates of the Intrinsic Ras GTPase hydrolysis in the presence of reactive oxygen species and reactive nitrogen species: HEPES, ph7.5, 298 K. Experiment In the presence of DTT (min -1 ) Control Nitric oxide (2X) xygen (2X) Nitric oxide (0.5X) & xygen (0.4X) Nitric oxide (0.5X) & xygen (0.2X) Nitric oxide (0.5X) & xygen (0.2X) In the absence of DTT (min -1 ) Impaired In the presence of the basal concentrations of the (0.5X) nitric oxide and (0.1X) oxygen, the intrinsic GTPase activity of the Ras protein is delayed by 3-4 times (Figure 3.28.b, Green). Results portray the inherent behavior of the GTPase protein in the presence of redox environment without precipitation. Further, the structural changes implied in the presence of nitric oxide and oxygen represents the primary changes before the protein undergoes any chemical reactions resulting in the degradation of the bound nucleotide and S-nitrosation of the Cys-118. In the presence of nitric oxide (0.5X) and oxygen (0.4X) there is no observable GTPase activity of the Ras protein, even though the Ras protein is not precipitated, but its GTPase activity is impaired under concentrations of nitric oxide (0.5X) and oxygen (0.4X) (Figure 3.28.b, Red). In the presence of excess concentration of nitric oxide and oxygen (data not shown), the Ras protein is completely precipitated without any valuable information and first few collected spectra represent a free nucleotide which is not bound to the Ras protein. Results also indicate, DTT has the capacity to regain the native GTPase activity of the Ras protein by inhibiting the formation of the reactive oxidation species. Experiments were further conducted to assess any modification of the bound nucleotide by HPLC in the presence of the low concentrations of the nitric ocide and oxygen from the FTIR sample (Appendix 8.12). From the results (Appenedix 8.12), which indicate that the flowthrough of the control Ras protein is identical to that of the Ras protein in the presence of the nitric oxide and oxygen [Nitric oxide (0.5X) & xygen (0.4X) or Nitric oxide (0.5X) & xygen (0.2X)]. The final conclusions from

122 110 the present study show small changes are associated with the Ras protein even in the presence of the low concentrations of the reactive redox species (Nitric oxide and oxygen), and also under these conditions, the bound nucleotide is not dissociated or undergone any chemical modifications Role of redox environment on the GTPase activity of the Ras protein in the presence of 1-2% NF1 Similar experiments were carried as in the Section with catalytic amount of NF1 to investigate whether GTPase activity is modulated. DTT free NF1 was obtained by forming complex with Ras caged GTP and fractions were collected through size exclusion process by a NAP-5 column. During sample preparation, Ras*cgGTP was taken in higher concentration to ensure excess Ras protein apart from Ras*cgGTP and NF1 complex. Photolysis and hydrolysis difference spectra are shown in the Figure 3.25.a, c. In the presence of nitric oxide and oxygen the characteristic 1689 cm -1 of Threonine 35 backbone vibration is lost and an increase in the intensity of γ phosphate at 1156 cm -1 was observed (Figure 3.25.a). The observed variations in the amide regions are due to presence of the three caged compounds and overlap between the reaction products from Ras*GTP, N and 2 respectively. Results indicate that in the presence of catalytic amounts of NF1, the GTPase of the Ras protein is delayed by 4 times in the presence of the (0.5X) nitric oxide and (0.2X) oxygen, while in the presence of the only nitric oxide there is no change in the GTPase activity (Figure 3.25.b). In the hydrolysis difference spectra there are not many changes associated with redox species in the phosphate regions, while as the amide I region of the Ras*GDP state is variable in nature, so any changes have no implications on the GTPase activity. In the phosphate regions, phosphate band at 1078 cm -1 is down shifted to 1076 cm -1 under the conditions of nitric oxide and oxygen (Figure 3.25.c). From the Figure 3.25.c, in addition to 1078 cm -1, there was also a red shift of 7 cm -1 in the region around 1386 cm -1 in the presence of the nitric oxide and oxide. The resulting red shifts at 1386 cm -1 and 1078 cm -1 were not observed in the case of the control protein or in the presence of the nitric oxide without oxygen.

123 111 Figure 3.25.a Photolysis difference spectra of Ras and 1-2% NF1 in the redox conditions: Ras Control, 1-2% NF1 (Black), Ras, 1-2% NF1 and nitric oxide (Red), Ras, 1-2% NF1, nitric oxide and oxygen (Blue), NPE caged GTP was used. Measurements were carried at 278 K, in HEPES, ph 7.7 in DTT free conditions. Absorbance Difference , cm sec sec sec log 10 time (Sec) Figure 3.25.b GTPase activity of Ras protein and 1-2% NF1 in the presence of redox enviroment at 278 K: Ras Control, NF1 (Black, 0.02 sec -1 ), Ras, NF1 and nitric oxide (Red sec -1 ), Ras, NF1, nitric oxide and oxygen (Blue, sec -1 ). Rates are fitted to first order exponential of the absorbance difference between the cm -1 w.r.t time.

124 112 Figure 3.25.c Hydrolysis difference spectra of Ras GTPase activity in the presence of the redox environment, Ras Control, NF1 (Black), Ras, NF1 and nitric oxide (Red), Ras, NF1, nitric oxide and oxygen (Blue). Measurement was carried at 278 K, in HEPES, ph 7.7 in DTT free conditions and scaled from 1236 to cm -1. The changes observed at the γ phosphate orientation (1156 cm -1 ) in the active state of the Ras*GTP is shown in the photolysis difference spectrum (Figure a) and after hydrolysis to Ras*GDP (Figure 3.25.c) whichh is observed with red shifts at 1386 cm -1 and 1078 cm -1 are the primary changes associated with the interaction of nitric oxide and oxygen. The changes observed in the spectra are without any precipitation of the proteins under study and there was no other side transformation of the bound nucleotide under these conditions. The final conclusions from these studies show that the GTPase activity is delayed depending on the concentration of nitric oxide and oxygen. Further there is no effect of the individual interactionn of the nitric oxide or oxygen on the GTPase activity. The results presented heree are for the first time elucidate the behavior of the Ras protein before it chemically undergoes any bound nucleotide dissociation or S-nitrosation or

125 113 oxidation at Cys-118, which is accomphlished at high concentrations of RNS or RS usually at high temperatures Interaction of the effector RalGDS with T35S in the presence of nitrosation environment In the present study, the behavior of the effector interaction with GTPase was studied under precipitation and non-precipitation conditions. For the experiments in the non precipitation conditions, the concentration of the nitric oxide was at half of that of proteins (RalGDS + T35S), while oxygen was maintained at one-fourth of the concentration of the RalGDS effector and T35S protein. ff state mutant RasT35S*GTP was selected for studying the behavior of the interaction of the effector with switch regions. The advantage of studying ff state mutant like T35S is that it gives clear information about the rate of the switching from ff state to n state only in the presence of the effector, while wild type switches from ff to n state without any requirement of an effector interaction. For precipitation conditions the concentration of nitric oxide and oxygen is at least 4 times higher than the concentration of the either proteins under study. The results indicate in the presence of lower concentration of the nitric oxide and oxygen, the effector RalGDS has interaction with the T35S ff state mutant, which is shown by the characteristic retention of the β Phosphate peak at 1217 cm -1 (Figure 3.26.a). The interaction of the effector with T35S is indicated by switching of the T35S mutant ff conformation to n conformation. The rate of the switching in the presence and in the absence of the nitric oxide and oxygen are very similar in the non precipitation conditions (Figure 3.26.b). The rate of switching during nonprecipitation conditions is 5.7 sec -1, while rate without nitric oxide and oxygen is 5.2 sec -1. The intensity of the γ phosphate vibration is reduced in the presence of the nitric oxide and oxygen mediated switching. Under higher concentration of the nitric oxide and oxygen, the effector still show the characteristic pattern of interaction, but the protein is completely precipitated. The rate under precipitation conditions is close to 8 sec -1, but information is lost for the first 200 msec as high concentration required higher number of laser flashes. In the precipitation conditions the β-phosphate band is not completely blue shifted to 1217 cm -1, but retained at 1214 cm -1 (Figure 3.26.a).

126 114 Figure 3.26.a Interaction of the effector RalGDS with Ras T35S*GTP in the presence of the nitric oxide and oxygen: photolysis difference spectra T35S and RalGDS (Black), T35S, RalGDS and low concentration of nitric oxide and oxygen (Blue), T35S, RalGDS and high concentration of nitric oxide and oxygen (Red), HEPES, ph 7.5, 258K & phpgtp was used, scaled at cm log 10 Time (Sec) Figure 3.26.b Kinetics of the generation of the normalized γ phosphate vibration of T35S at 1144 cm -1 in the presence of the effector RalGDS. control, higher concentration of the nitric oxide and oxygen, low concentration of the nitric oxide and oxygen.

127 115 The final conclusion from this results indicate that nitric oxide mediated free radical activation of Ras protein Cys-118 has no role in the recognition of the effector RalGDS by the switch regions of the Ras protein. Conversely, it could be concluded that milli second events like protein-protein interactions are not perturbed by nitric oxide mediated nucleotide oxidation, which is a process usually accomplished in the range of the minutes, also temperature play an important role in such oxidation process. The choice of RalGDS which has least affinity to the Ras protein compared to the affinity of RafRBD for the Ras protein, implies that the other effectors which have similar domain recognition on the Ras protein for the RalGDS, might behave in a similar fashion in the presence of nitric oxide and oxygen Anticancer activity of Ruthenium nitrosyl complex The potential of the Ruthenium nitroso complex (RuA) as an anticancer agent was examined employing cytotoxic studies on HT29 cell lines by Resazurin (Alamar Blue) assay and Crystal Violet assay. The UV fluorescence results from the Resazurin (Alamar Blue) assay and Crystal Violet assay are shown in the Figure 3.27 a & b respectively. Resazurin assay measures the fluorescence increase of the Resorufin (pink) from the mitochondrial metabolic activity of the living cells on the nonfluorescent Resazurin (Blue) ('Brien, J. et al., 2000). Under controlled conditions, the results indicate, that in comparison to Sodium nitroprussade (SNP), a common nitric oxide donor, Ruthenium Nitrosyl complex is much more effective in promoting cell death under various time durations of UV light irradiation. In both cases of metal nitric oxide donors, the survival of the cells before UV irradiation is comparable, but shows opposite effect in the presence of the UV light. At low concentration (0.1mM) ruthenium nitroso complex (RuA), the efficiency is around 60%, while SNP has less than 10% mortality rate. The probable reason is the stability of the Ruthenium nitrosyl complex (RuA) in water and to normal light, while SNP is not stable to either. ther control experiments with 5-flurouracil proves that Nitroso Ruthenium complex shows the activity based on UV light exposure rather than transcription inhibition initiated by 5-flurouracil (5FU). Ruthenium complex without nitric oxide (RuB) has no comparable activity initiated by UV irradiation. However, no influence on the growth characteristics caused by the presence of the organic solvent (5% DMS) was observed and also mortality rate of cells exposed to UV is less than 10 % indicating that cell death is cuased by the nitric oxide released from metal nitrosyl complex.

128 Min 10 Min 25 Min Percentage SNP SNP RuA RuA RuB RuB 5UF Cell Cell DMS 1mM 0.1mM 1mM 0.1mM 1mM 0.1mM 0.02mM NoUV UV 5% Figure 3.27.a Resazurin (Alamar Blue) assay: The percentage survival of the HT29 colon cancer cells under UV irradiation at 365nm. The conditions are labeled with the compounds SNP (sodium nitro prussade) (1mM, 0.1mM), RuA (Ruthenium nitroso complex) (1mM, 0.1mM), RuB (Ruthenium chloro complex), 5-Flurouracil (0.02mM). Each experiment is done on triplicate scale and values are averaged. The differential role of SNP and ruthenium nitrosyl complexes in cell mortality is due to the stability of the latter metal complex, which increase the local concentration of the nitric oxide during UV irradiation and GTPases are inhibited in the presence of higher concentrations of nitric oxide and oxygen. In the Crystalviolet assay (Figure 3.27.b), chemosensitivity is quantified by staining cells with crystal violet in the estimation of the viable biomass (Bernhardt, G, et al., 1992). Figure 3.27.b summarizes the effect of Ruthenium nitroso complex on UV irradiation. The results observed with Resazurin assay are also observed in the crystal violet assay. The repetition of the similar results from both assays demonstrates that the Ruthenium Nitrosyl complexes have a potential in

129 117 the treatment of the cancer showing ruthenium nitroso complex as an efficient chemical drug only under the influence of UV light irradiation Min 10 Min 25 Min Percentage SNP SNP RuA RuA RuB RuB 5UF Cell Cell 1mM 0.1mM 1mM 0.1mM 1mM 0.1mM 0.02mM NoUV UV Figure 3.27.b Crystal Violet assay: The percentage cell mass recovered from the intact HT29 colon cancer cells after UV irradiation at 365nm.cell. The conditions are labeled with the compounds SNP (sodium nitroprussade) (1mM, 0.1mM), RuA (Ruthenium nitroso complex) (1mM, 0.1mM), RuB (Ruthenium chloro complex), 5-Flurouracil (0.02mM). Each experiment was done on a triplicate scale and values are averaged. Application of such compounds help in the development of therapeutics in the treatment of cancer in a well defined area only under UV light irradiation rather than exposing the normal and healthy cells. Light as an initiator of toxicity helps in preventing the toxicity to normal cells compared to the cancer cells which are exposed to light.

130 118 4 Discussion 4.1 Semisynthetic Ras Protein Synthesis of site specific labeled proteins by Semisynthetic technique is a challenging task. The two samples of the semisynthetic Ras protein provided by Dr. Christian Becker, MPI, Dortmund, have different physical properties in terms of the molecular weight (Appendices 8.4.a,b). Measurements with correct molecular mass semisynthetic Ras protein (18.8 kda) revealed a similar photolysis behavior like that of wildtype protein with minor changes in the β- and γ- phosphate intensities and associated with small band shifts compared to the Ras wild type protein. There were also minor changes observed in the intensities of the amide-i regions and while refolded Ras wild type protein has the inherent RasGAP assisted GTPase activity, but the semisynthetic protein was not active. Inactivity of the semisynthetic Ras protein in terms of its protein bound GTP hydrolysis is a serious problem in continuing this project further. It is surprising, that despite the similar photolysis spectrum, no hydrolysis can be obtained, probably due to small folding defects. 4.2 Switching of conformations with small molecules Switching of Ras protein from ff state to n state is an observable property with well defined kinetics and IR frequencies. The modulation of the switching process in favor of the ff conformation seems to be an important strategy in muting the Ras mediated signal transduction as ff conformation has weaker affinity for the effectors. trftir studies show interaction of zinc cyclen with Ras*GTP has no influence on the modulation or equilibrium delay of the conformation states as described by NMR studies which were conducted on Ras*GppNHp (Spoerner, M., et al., 2005). Present results from trftir studies indicate the conclusions from NMR regarding the modulation of the Ras protein with zinc cyclen seems to be a speculation and it might represent a process describing a nucleotide dependent artifact. As recent studies by NMR revealed the existence of only one conformation state without any observable populated ff state, when Ras is bound to GTPγS and also there are no observable other sub-states which are populated (Spoerner, M., et al., 2005a). In trftir studies, the caged GTP imposes the protein into an induced ff conformation state before photolysis. For

131 119 inherent n state conformation proteins like Ras wild type, G12V & Q61A, there is a tendency to attain n state after the decaging of the nucleotide by photolysis. The transition from ff state to n state is followed with a rate close to 5 sec -1 at 260 K. While ff state mutants like T35S, M-Ras, G60A (exception Y32W) all lacks coordination of γ-phosphate to Threonine 35 backbone resulting in an open conformation (Figure 4.1.a). Switch II Switch I Possible Anti-Cancer Conformations Figure 4.1.a Comparison of possible conformations for Anti cancer strategies: Superimposed crystal structures of Ras*GTP wild type (1QRA, green), Ras wild type (GDP, AlFx complex) in association with GAP protein in the transition state (1WQ1, hot pink), GAP is removed for clarity. M-Ras*GppNHp (1X1S, Golden orange), Ras*cgGTP (1PLJ, Yellow), G60A*GppNHp (1ACM, Blue). The anticancer strategies are only possible for the Ras wildtype with GTP (Green) and cggtp (Yellow) as the bound nucleotides. Switch regions are indicated, and + is a water molecule. Interaction of RafRBD with ff state mutants bound to GTP (exception T35A) results in the closing of the switch regions to attain a closed n state conformation with a unique kinetic rate. The results indicate that ff state mutants bound to GTP also lack the ability to interact with the zinc cyclen in the presence of the RafRBD indicating there is probably no interaction of the zinc cyclen close to phosphate moiety bound to the protein as described by NMR studies, which were done on Ras bound GppNHp (Spoerner, M., et al, 2005).

132 120 The ability to modulate the switch regions were probed with a couple of compounds other than zinc cyclen including Spermidine, Spermine, Putrescene and 1,2,3-Hexanetriol. All of these compounds have no effect on the Ras modulation in the presence of the equimolar Mg 2+, while at low concentrations of Mg 2+, they showed the tendencies to dissociate the nucleotide by chelating with the cofactor Mg 2+ ion. To probe the volume and surface change associated with Switching mechanism, experiments were conducted similar to MSCS (MultiSolvent Crystal Structures) method (Mattos, C., et al., 2003). The use of organic solvents in the MSCS method is shown to induce stability of the switch regions especially switch II motif. In the present study, DMS and Acetonitrile are shown to interact with switch regions, which delayed the rate of switching from ff conformation to n conformation by about 5 times, with the final n state retaining the proper phosphate regions in the FTIR spectrum. The results from applied MSCS method into trtfir studies imply that the surface and volume change of the Ras wild type protein is very minute as DMS and Acetonitrile have a very small surface area. From the inspection of the crystal structures of the Ras protein bound to caged nucleotide (PDB: 1PLJ) the sum changes associated with switch regions are very minute and the distances between side chain of the Threonine 35 in n state (PDB: 1QRA) and ff state (PDB: 1PLJ) is less than 1.8 Å (Figure 4.1.b). The characteristic Tyrosine 32 residue in the n state is shown to envelop the phosphate regions, while in the ff state backbone (PDB: 1PLJ) is shown to be slightly shifted outside by about 2.2 Å retaining its spacial orientation as that of n state (Figure 4.1.b). The side chain of the Tyrosine 32 namely the aromatic ring undergoes a considerable change during switching from ff state to n state, the hydroxyl on the aromatic ring transverse about a distance of 6.3 Å during switching (Figure 4.1.b). The residues on the Switch II region are not that much perturbed during switching from ff to n state, namely Glycine 60, which has less than 0.4 Å movement during switching from ff to n state. n switching from ff state to n state, the volume change of such a process with respect to n state (PDB: 1QRA) transition to ff state (PDB: 1PLJ) or vice versa with respect to Mg 2+, backbone movement of the essential residues like Threonine 35 and Tyrosine 32 is less than 12.0 Å 3 (Figure 4.1.b). The molecular entities which are suitable to fit into such a volume between the switch regions are not larger than a single carbon-carbon bond. Present results with DMS and

133 121 Acetonitrile confirm the existence of such a scenario which is shown to delay the switching kinetics by 5 times (Figure 3.8). Figure 4.1.b Switch movement from ff State to n State, verlay of the Ras*GTP (PDB :1QRA, Green) and Ras*cgGTP (PDB:1PLJ, Yellow). Switch regions are indicated with essential residues shown in stick models, Mg 2+ is shown in hot pink The other ff conformation states recently published for M-Ras (PDB: 1X1S) and Ras G60A (PDB: 1ACM) are shown to exist in well defined ff state conformation in which switch regions are open, especially threonine 35 has no coordination to γ phosphate and magnesium (Ye, M., et al, 2005 ; Ford, B., et al, 2005). In the case of M-Ras protein, there is a vital mutation at the Glutamic acid 30 residue to proline, which abolish the essential hydrogen bonding between the nucleotide and Switch I region. Also the presence of two proline in less than three residues apart in the Switch I regions imparts a stiff conformation of the switch I loop resulting in an ff state (Ye, M., et al, 2005). From the inspection of the overlap of all ff state mutants (MRas, Ras G60A) crystal structures with respect to n state Ras wild type shows anticipated deviations in the switch regions (Figure 4.1.a). The structures of G12V and Q61A have similar switch orientations

134 122 compared to that of Ras wild type and also similar FTIR photolysis difference spectra. The mode of modulation of the switch regions in G12V and Q61A should be similar to the wild type. The possible volume change of the G12V and Q61A should also be less than 12.0 Å 3 as described above for wild type (PDB: 1QRA, 1PLJ). The possible anticancer strategy involving modulation of the switch regions should target by the drugs which could fit the volume less than 12.0 Å 3 (Figure 4.1.b). The forthcoming conclusion from the anticancer strategy shows the prominent structure which is important for cancer treatment should be PDB: 1PLJ which is shown in yellow color in the Figure 4.1.a is a caged GTP bound to Ras protein and all other ff state mutants (M-Ras, G60A) are not applicable in targeting H-Ras therapy. 4.3 Ab initio studies and implications of non-hydrolyzable nucleotides The Ab initio studies on the T1, T2 and T3 model compounds give an indication about the influence of the delocalization of the lone pair electrons on the bridging atom of the triphosphate model behavior. These variations are clearly visible in the nature of the bond lengths connecting the bridged β,γ-phosphates which indicates an increase of 0.25 Å, while intense frequency of T1 is a coupled vibration, the models of T2, T3 demonstrate a behavior with strongest vibrations arising from the bridging atom bending and wagging motion out of plane with respect to the bonds of NH and CH 2. This sort of vibration is not present when oxygen is present as a bridging atom, as symmetry forbids this bending motion. The assignments of the frequencies due to specific groups can be quantitatively made from ab initio frequency evaluation, which can aid in better understanding of experimental results (Förner, W., et al., 2000). The implications from the ab initio studies of T1, T2, and T3 can be applied to interpret the experimental results in a qualitative manner. The results from trftir studies reveal the vibrations from the phosphate regions of the Ras bound GppNHp and GppCH 2 p show equilibrium shift, which are dependent on the temperature. Further all the positive peaks ranging from cm -1 analyzed from the trftir of Ras*GppNHp and Ras*GppCH 2 p have no well defined kinetic behavior compared to that of Ras bound GTP, while only peak at 1614 cm -1 showed similar kinetic behavior for Ras*GTP and Ras*GppCH 2 p. Interaction of the zinc cyclen with Ras*GppCH 2 p has shown considerable perturbation of the switch regions, implies the switch regions are quite fluxional in the presence of metal phosphate chelate like Zinc cyclen. While Ras bound GTP doesn t show any fluxional changes of the γ-phosphate intensity with variation in temperature or with

135 123 interaction of the Zinc cyclen. The existence of population levels of State 1 and State 2 as described by NMR are due to fluxional nature of GppNHp and GppCH 2 p, where γ-phosphate is clearly perturbed by the NH and CH 2 wagging motion. The analysis of the distances between the phosphate groups from the published crystal structures of the GTPases are tabulated in the Appendix 8.4. The analysis of the distances between pair of the phosphate centers from GTP, GppNHp and GppCH 2 p, show that in the protein bound GTP form, the distances between the pair of γ-, β phosphates and β-, α phosphates in most cases are nonequivalent. n the other hand, GppNHp and GppCH 2 p bound protein display equivalent distances between the pair of the phosphate centers. The analysis of the distances between pair of phosphate centers shows nonequivalent environment around the nucleotide binding domain depending upon the type of the nucleotide. The convincing example could be given from the analysis of the RhoC protein crystal structures (Dias, S.M., et al, 2007), for this protein depending on the type of the nucleotide bound, different conformation states are observed. In GTPγS bound state, RhoC is always found to be in predominate n conformation state, while with GppNHp or GppCH 2 p bound state, protein is observed with ff conformation with open switch regions, which indicate the tendency of the switch regions to interact with γ phosphate depends up on the nature of the bound nucleotide. The possible alternate explanation for nonequivalence of the nucleotide states could be given from the comparison of the pk a between GTP, GppNHp, and GppCH 2 p. For the free nucleotides in the solution, the pka values for the γ-phosphate group increase from 6.3/4.7 for GTP/GTP*Mg 2+ to 8.9/6.3 for GppNHp/ GppNHp*Mg 2+ and 9.0/6.6 for GppCH 2 p/gppch 2 p*mg 2+ respectively (Spoerner, M., et al., 2005a). The change in the pk a values make the γ phosphate of GppNHp and GppCH 2 p less inherent to act as H-bonding acceptor for the switch residues like Threonine 35, and Tyrosine 32, which possibly explain the dynamic existence of the conformation state equilibria from NMR studies on Ras*GppNHp and Ras*GppCH 2 p. 4.4 GTPase activities with small molecules and metal NF1 peptide biocojugates From the present study, results indicate that metal macrocycles have an effect on the rate of the hydrolysis of the free GTP hydrolysis. Susbstition of the cyclam derivatives increased the rate of

136 124 the hydrolysis by 10 times for L1 series of the metal macrocycles, while for L2 series the rate of free GTP hydrolysis was close to times compared to free GTP without any metal macrocycle. Metal derivatives of the Fab series of the ligands also showed increase in free GTP hydrolysis rate by 25 times. The interaction of the metal derivatives of cyclam of L1, L2 and Fab series of compounds with protein bound GTP has no effect on the rate of the Ras wild type and G12V bound GTP hydrolysis rate. The results indicate, the substitution derivatives of the cyclam has ability to accelerate the free GTP hydrolysis as studied by HPLC, but they don t have the ability to orient functional groups similar to a biological enzyme or a protein domain like GTPase activating protein (GAP) to accelerate Ras bound GTP hydrolysis. The success of the downregulation of oncogenic Ras protein from the previous studies (Ahmadian, M.R., et al., 1999 &, Gail, R., et al., 2001) where acceleration of the protein bound nucleotide was accomplished by supplying a catalytically functional group into the active site of oncogenic Ras proteins to improve their primary biochemical defects. The results from such studies revealed a process of aminolysis rather than inherent mechanism for the observed increase in the GTPase activity, here the results with substituted macrocyles of L2 from the present study have not shown any effect similar to DABP-GTP analog even though having an amine side chain like that of DABP-GTP. Further studies were conducted employing the nature s principle of selection in accelerating the hydrolysis of Ras bound GTP. The selection of the catalytic sequence was from RasGAP protein which has an essential Arg residue known as Arginine finger. The peptide sequence was conjugated with metal macrocyle for effective coordination to the carboxylates of the protein by the metal center. Effort in synthesizing the Ruthenium NF1 peptide bioconjugate on first trial has succeeded, though the resulting metal bioconjugates constituted a mixture of the required bioconjugate and also with minor fraction of N-truncated NF1 peptide conjugates, with one and two missing linking glycine residues from the N-terminus of the NF1 peptide catalytic sequence Gly-Gly-Gly-Thr-Leu-Phe-Arg 789 -Ala-Thr-Thr-Leu-Ser-Arg. Apart from the synthesis the main focus of the study being the GTPase acceleration with metal peptide bioconjugates. The present results show, the interaction of the Ruthenium bioconjugate with Ras*GTP delays the rate of hydrolysis by times at 303 K. The interaction of the metal complex with Ras protein has no effect on the rate of the Ras GTPase activity, but the interaction of Ras protein with metal peptide bioconjugate shows a net delay in the rate of the GTPase activity. The indications are

137 125 likely to show that mere presence of sequence from the NF1 catalytic Arginine finger 789 and associated loop sequence from NF1 is not enough to ascertain the acceleration of the GTPase activity. The transition state structure from GDP*AlF x complex of Ras and NF1 indicates phenomenal changes are associated with RasGAP interaction and activation of Ras GTPase activity in the switch II region (Figure 4.2). Compared to Ras*GTP (PDB: 1QRA, Green), the most changes in transition state structure of the GAP complex (PDB: 1WQ1, Hot pink) are associated with the movement of the Switch II regions shown in golden orange color, while Switch I regions (blue color) are quite stationary with respect to uncomplexed protein state, even after the cleavage of the γ-phosphate. The present attempt of employing Ruthenium NF1 peptide bioconjugate to accelerate the GTPase activity is not conclusive; and there are no reasonable explanations why the influence of this metal peptide bioconjugate inhibits the intrinsic GTPase activity, but at least there seems to be a complex formation. Figure 4.2 Switch Movement: Superimposed crystal structures of Ras*GTP wild type (1QRA, green) and GDP*AlFx complex of Ras wild type in association with GAP protein (1WQ1, hot pink), GAP is removed for clarity. Blue regions are switch I residues, while Golden orange regions correspond to Switch II residues, which show significant movement on association with GAP protein.

138 126 There are minute changes associated with interaction of Ruthenium NF1 peptide bioconjugate with Ras wild type protein in the presence of 1% NF1 at the initial states of the photolysis, while interaction of 1:1 complex of NF1 protein with Ras wild type protein progress through a rapid rearrangement of the switch II regions (PDB 1WQ1, Figure 4.2), which is not possible with mere association of peptide loop sequence of NF1 as studied here. The possible reason being in the transition state of the GAP complex (PDB: 1WQ1), there is a helix:helix interaction between the switch II region of the Ras protein and a helix domain of the GAP protein resulting in the conformation changes of the switch II regions with reorientation of the critical Glutamine 61 of the Ras protein. The changes in the amide regions of hydrolysis spectra of the 1% NF1 assisted GTPase activity in the presence of Ruthenium NF1 peptide bioconjugates are not surprising, as final GDP state is known to have multiple conformations due to flexible switch regions. As expected, the inability to hydrolyze the GTP bound to oncogenic Q61A protein by Ruthenium NF1 peptide bioconjugate presents a complex phenomenon by which GAP proteins function to accelerate the protein bound GTP hydrolysis. For example, RapGAP uses Asparagine as catalytic residue without any transition state complex (Daumke,., et al., 2004), while RasGAP uses Arginine in the stabilization of the transition state of protein and with observable intermediate state during GTP hydrolysis (Ahmadian, M.R., et al., 1999, Allin, C., et al., 2001a). Previous studies involving the emulating of the active sites (Berkessel, A., et al., 1999) of the proteins with synthetic peptides corresponding to ubiquitous sequences surrounding the phosphate moiety of the proteins to accelerate the phosphate hydrolysis had very limited success. The library screened from such studies involved 652 peptide 12mer sequences and of which only three sequences showed activity of the interest which moderate improvement. ne of the inactive sequences studied from that library is similar to the present synthesized Ruthenium NF1 peptide bioconjugate. Even the active sequence from that study does not necessitate the requirement of the Arginine as a critical residue in order to enhance the phosphate hydrolysis. This preference could also be seen in Rap1 and its associated GAP namely RapGAP, which harness helix comprising the Asparagine 290 as a critical residue instead of Arginine to enhance the protein bound GTP hydrolysis rate. Compared to NF1 or RasGAP, RapGAP has a tendency to downregulate the RapG12V mutation. Alternative strategies may include the screening the sequences of the RapGAP which consists of helices harboring Asparagine as the critical residue in the library sequences.

139 Nitrosation induced changes and associated down-regulation of Ras protein From the recent developments on the studies of nitric oxide cell signaling and its implication of S-nitrosation of Ras superfamily GTPases modulation (Raines, K.W. et al., 2007), a considerable attention is targeted at understanding the mechanism of nitric oxide mediated nucleotide exchange of Ras protein. Results from previous studies on the Ras GTPase indicate that N can promote formation of a cysteine thiol radical (Cys118), which alters Ras activity through a radical propagation mechanism leading to guanine nucleotide oxidation and release of GDP from Ras protein. However, the final state of the reaction in which Cysteine undergoes S-nitrosation at the residue 118, this S-nitrosylated cysteine (Cys118-SN) does not effect Ras biochemical activity or structural integrity. Hence, the mechanism of N-mediated regulation of Ras activity is distinct from what has previously been observed in other systems, in which protein S- Nitrosation or S-Nitrosylation alters cellular function. In the present study, the role of formation of the cysteine thiol radical on the Ras GTPase activity and interaction of RalGDS are probed under nonprecipitation conditions. Nitric oxide has a tendency to activate or inactivate the small GTPases depending on the level of the N concentration. Under higher concentrations, N signaling in the cell results in small GTPases inhibition, while at low concentration it shows the tendency of activation of the small GTPases (Raines, K.W., et al., 2007). Former property of N can be used as an effective anticancer strategy. Under controlled nonprecipitation conditions results from the present study demonstrate, the GTPase activity of the Ras protein is delayed around 3-4 times in the presence of low concentrations of nitric oxide (0.5X) and oxygen (0.2X). While in the presence of excess nitric oxide, the protein is practically precipitated without any observable GTPase activity or any of the characteristic Ras*GTP spectrum. In the case of the interaction of RalGDS with ff state mutant like T35S, results indicate that T35S has a tendency to interact with effector RalGDS in the presence of nitric oxide and oxygen. The switching rate of T35S and RalGDS is not altered under lower concentrations of nitric oxide and oxygen, while under higher concentrations of nitric oxide the protein is precipitated but retained its ability to interact with RalGDS. The interaction of the precipitated protein with the effectors was also observed with NMR studies as previously reported (Ader, C., et al., 2007, & Iuga, A., et al., 2006), which show GTPases have normal behavior in challenging conditions like precipitation with ammonium sulfate or PEG

140 The kinetic rate was also not altered in the presence of higher concentrations of nitric oxide and oxygen, but it is observed with a red shift of β-phosphate vibration on interaction with effector RalGDS, compared to normal interaction of RalGDS without any nitric oxide and oxygen. The present study demonstrates that in the presence of the various concentrations of the nitric oxide and oxygen, the effector has an inherent tendency to interact with switch I region of the Ras protein. The possible outcome from this study shows that nitric oxide initiated free radical propagation has less influence on the millisecond events of the protein-protein interactions between Ras protein and its effector RalGDS. In vitro studies of Ras nucleotide dissociation in the presence of the Ruthenium nitrosyl complex and titanium peroxo citrate is accomplished by application of UV irradiation at 365nm. The photolysis of nitric oxide from Ruthenium nitrosyl complex and oxygen from titanium peroxo citrate resulted in the formation of Reactive Nitric xide Species (RNS) and Reactive xygen Species (RS). The Mass spectral analysis of the flowthrough from the in vitro studies by the RNS/RS induced Ras nucleotide dissociation resulted in 5-Nitro and 5-xo species of the respective protein bound nucleotide (Appendix 8.11). Further, the presence of metal macrocycles also cleaved the phosphate moieties from oxidized nucleotides, which are identified by mass spectra. Results from cytotoxic studies on HT29 colon cancer cell lines indicate that Ruthenium Nitrosyl complex (RuA) is effective in killing 60% of the cells in a controlled fashion under the influence of UV light irradiation. Similar studies with sodium nitroprussade showed the opposite effect. The possible reason is the instability of nitric oxide complex of Iron (SNP), and during the incubation period about 48 hrs after the addition of the compounds, most of the nitric oxide is dissociated from the SNP, reducing the effective concentration of the nitric oxide before UV light illumination, while Ruthenium Nitrosyl complexes are very stable and only undergo decomposition only in the presence of UV light illumination. The Ruthenium complex without any nitric oxide has no ability to induce cell death by UV light illumination and also Ruthenium Nitrosyl complexes has a similar ability like 5-Flurouracil as an anticancer agent. The advantage of Ruthenium Nitrosyl complexes is its ability to harness the potential of decaging the nitric oxide under UV illumination giving a choice in targeting the certain regions of interest for the treatment of cancer.

141 129 In conclusion, the results from the present studies demonstrate a controlled ability to send a death signal to specific cell regions under UV light irradiation at 365nm on Ruthenium Nitrosyl complexes. The possibility of a control in killing the cell in a desired fashion is of utmost importance in the cancer therapeutics for which Ruthenium nitrosyl complexes can be classified as an effective anticancer therapeutic. 4.6 Future Prespectives In the continuation of the success in the present study, the results could be employed in the development of new class of Ruthenium anticancer agents not only harnessing the Nitric xide derived substitutents of the ruthenium but also DNA chelating capacity of the Ruthenium macrocycles as shown with ligand Z in black spheres in the Figure 4.3 (Pascu, G. I., et al., 2007). Z [Ru 2 Z 3 ] 4+ Figure 4.3 Structure of Z and [Ru 2 Z 3 ] 4+ cation. Ru large black spheres, N small black spheres, C small gray spheres. Hydrogen atoms, anions, and cocrystallized solvent molecules are omitted for clarity (Pascu, G. I., et al., 2007).

142 130 The shown Ruthenium complex is found to bind DNA and exhibit anticancer activity. Also effort should be made in development of Ruthenium nitrosyl complexes with higher quantum yields. From the present study, results of Ras*GTP hydrolysis rate shown to be delayed in the presence of nitrogen oxides, it is very interesting to find whether such a process is also observed with RasGAP assisted Ras*GTP hydrolysis and also information about intermediate behavior like reversibility or rate determining steps in the presence of the nitrogen oxides. It is also interesting to investigate whether GAP protein can sense the difference between the redox active GTPase like Ras wild type and also redox deficient mutants like C118S & F28L which are known to be insensitive to N mediated modulation.

143 131 5 Summary The aim of this work is the downregulation of the Ras protein by metal macrocycles, as commonly involved mutations in Ras protein result in cancer. That Ras*GTP bound form is an attractive therapeutic target seems to arise from its multiple pathway involvement in signal transduction. Impeding Ras*GTP protein association with any of the effectors mutes the signal transduction, and is a promising phenomenon in the prevention of the cancer. Here the following approaches were investigated on the Ras protein. A) Switching mechanism B) GTP hydrolysis acceleration C) Metal NF1 bioconjugate GTPase Activating Protein mimics D) Photo Dynamic Therapeutic applications in nucleotide dissociation A) Ras protein is observed with transition from ff state to n state in the GTP bound form. Modulation of conformation in favor of the ff state provides a handle to control the effectors interaction with GTP bound Ras protein. Modulation of Ras protein with a phosphate chelating agent like Zinc Cyclen is a novel mechanism as probed by NMR studies with Ras*GppNHp. This phenomenon as elucidated by NMR studies is an observable nucleotide dependent artifact, as results from trftir showed no observable changes associated with Ras conformational switching between ff and n states using GTP as the bound nucleotide. Further, the ff state mutants like T35S and Y32W which prefers to adapt a predominant ff conformation are switched back to n State in the presence of RafRBD and the zinc cyclen. The present results are in conformity with crystal structures from Ras bound GTP (1QRA, 1PLJ) with respect to the available free volume between n and ff state. The free volume criteria was further confirmed with effect of organic solvents on switching kinetics, which show that the volume occupancy for such a surface change during switching process can accommodate molecule not larger than 12 Å 3. Theoretical and experimental studies on nonhydrolyzable nucleotides revealed the frequencies are downshifted and bond orders are different for γ-phosphate of the GppCH 2 p compared to GTP. trftir studies on interaction of Zinc cyclen with the Ras*GppCH 2 p resulted in fluxional behavior of the switch regions which was not observed with Ras*GTP.

144 132 B) Substituted cyclam derivatives showed acceleration of the free GTP hydrolysis, while these compounds have no effect on the protein bound nucleotide hydrolysis rate. C) Acceleration of Ras bound GTP hydrolysis is an alternative mechanism in the downregulation of the Ras signaling pathway. GTPase Activating Proteins (GAPs) are negative effectors of Ras signaling, which accelerate the GTP hydrolysis by 10 5 fold. Mimicking the native GAP activity with a chemically synthesized moiety is a challenging process. Here ruthenium macrocycle is conjugated with peptide sequence of NF1 by incorporating the essential Arg789 namely Arginine Finger. The interaction of resultant metal conjugate with Ras wildtype reduced the GTP hydrolysis rate by times intrinsically at 303 K, while with 1% NF1 assisted GTP hydrolysis, the metal conjugate shows a deceleration by 10 times. The resulting studies of oncogenic mutant namely Q61A with metal conjugate has shown no improvement in GTP hydrolysis. D) Increasing the nucleotide dissociation is also an alternative mechanism of Ras downregulation. Compounds which serve this phenomenon could be important tools in cancer treatment. Ruthenium complexes have been shown to exhibit promising anticancer activity in vitro and in vivo and these compounds thus provide a very attractive alternative to cisplatin. The aim of this project is the application of photo-labile metal complexes. Nitrosyl ruthenium complexes are shown to have a higher activity, a lower general toxicity and new modes of action compared to other forms of nitrosyl complexes. Nitrosation in the presence of oxidizing RNS or RS on the Ras protein namely at the residue C118 results a nucleotide free apo state. Cell based assays showed that ruthenium nitrosyl complexes inhibit cancer progression specifically by around 60% under controlled conditions compared to other forms of nitrosyl complexes. Apart from its anticancer activity, the role of the nitrosylation is investigated in terms of GTPase activity. Under controlled conditions, intrinsic GTPase activity is delayed in the presence of the RNS and RS species from Ruthenium Nitrosyl complexes and also protein interaction between the effector proteins and GTPase are not influenced by RNS and RS. Further, provided semisynthetic Ras protein from MPI, Dortmund, is not active in terms of its GAP assisted Ras bound GTP hydrolysis.

145 133 6 Zusammenfassung Das Ziel dieser Arbeit ist die Herunterregulation des Ras-Proteins durch Metallmakrozyklen, da Mutationen im Ras häufig eine Ursache für die Entstehung von Krebs darstellen. Zu seiner Attraktivität als therapeutisches Ziel trägt die Tatsache bei, dass Ras an mehreren Signaltransduktionswegen beteiligt ist. Eine Störung der Verbindung von Ras mit seiner Effektoren unterdrückt die Signaltransduktion und ist daher ein vielversprechender Ansatz, der zur Verhinderung von Krebs eingesetzt werden könnte. Hier wurden die Folgenden Annäherungen von Ras mit gebundenem GTP zu erreichen. A) Umschaltmechanismus B) GTP Hydrolyse Beschleunigung C) Metall-Biokonjugat GTPase Aactivating Protein Imitate D) Photo Dynamic Therapy Anwendungen in der Nukleotiddissoziation Ras in der GTP gebundenen form kann beim Wechsel Ubergang vom "ff" in den n Zustand beobachtet werden. Die Modulation der Konformation in einer den "ff" Zustand begünstigenden Weise, ist ein Ansatzpunkt zur Kontrolle der Wechselwirkung von Effektoren mit aktiviertem Ras. Die Modulation von Ras mit phosphatchelatisierenden Reagenzien wie Zink-1,4,7,10-Tetraazacyclododekan stellt laut NMR-Studien mit Ras*GppNHp einen neuartigen Mechanismus dar. Dieses Phänomen ist ein Nukleotid abhängiges Artefakt in den NMR-Studien, da in trftir-messungen keine Veränderungen bezüglich eines konformationellen Umschaltens zwischen dem ff und n Zustand auftraten. Des Weiteren werden ff Zustand Mutanten wie T35S und Y32W, die vorwiegend eine ff Konformation einnehmen, in Gegenwart von RafRBD und Zink-1,4,7,10-Tetraazacyclododekan wieder in den n Zustand versetzt. Die gegenwärtigen Ergebnisse stehen im Einklang mit Kristallstrukturen von Ras gebundenem GTP (1QRA, 1PLJ) in Bezug auf den verfügbaren freien Band zwischen n und ff Konformation. Die freien Band Kriterien wurden zusätzlich durch den Effekt organischer Lösungsmittel auf die Umschaltkinetik bestätigt, Theoretische und experimentelle Untersuchungen von nicht hydrolisierbaren Nukleotiden zeigten, dass für γ-phosphat die Frequenzen zu niedrigeren Werten hin verschoben und die Bindungsordnungen verschieden sind.

146 134 trftir-untersuchungen der Interaktion von Zink-1,4,7,10-Tetraazacyclododekan mit Ras- GppCH 2 p zeigten die flexible Natur der Switch-Bereiche, die bei Ras-GTP nicht beobachtet wurde. B) Substituierte Cyclam-Derivate riefen eine Beschleunigung der Hydrolyse freien GTPs hervor, während diese Verbindungen keine Wirkung auf die Hydrolyse-Rate des proteingebundenen Nukleotids hatten. C) Die Beschleunigung der Hydrolyse Ras gebundenen GTPs ist ein alternativer Mechanismus zur Herunterregulation des Ras-Signaltransduktionsweges. Aktivierende Proteine (GAPs) sind negative Effektoren der Ras-Signaltransduktion, die die GTP Hydrolyse um den Faktor 10 5 beschleunigen. Das Imitieren der nativen GAP-Aktivität durch eine chemisch synthetisierte Verbindung stellt eine Herausforderung dar. Hier wird ein Ruthenium-Makrozyklus mit der Peptidsequenz von NF1 konjugiert, die den essentiellen Rest Arg789 d.h. den sogenannten Argininfinger beinhaltet. Die Wechselwirkung des resultierenden Metallkonjugats mit der Wildtyp Form von Ras reduzierte die intrinsische GTP Hydrolyse-Rate um den Faktor 15, während das Metallkonjugat bei einer mit 1% NF1 unterstützten GTP Hydrolyse eine Verlangsamung um den Faktor 10 hervorrief. Die resultierenden Untersuchungen der onkogenen Mutante Q61A mit dem Metallkonjugat zeigten keine Verbesserung der GTP Hydrolyse. D) Die Erhöhung der Nukleotiddissoziation ist ein weiterer Mechanismus zur Herunterregulation von Ras. Verbindungen, die diesen Effekt hervorrufen, könnten wichtige Hilfsmittel in der Krebstherapie sein. Es wurde bereits gezeigt, dass Rutheniumkomplexe in vitro und in vivo eine vielversprechende Antikrebs-Aktivität aufweisen und daher eine sehr attraktive Alternative zu Cisplatin darstellen. Das Ziel dieses Projektes ist die Anwendung von photolabilen Metallkomplexen. Nitrosyl- Ruthenium-Komplexe haben eine höhere Aktivität, eine niedrigere allgemeine Toxizität und zeigen neue Wirkungsmechanismen im Vergleich zu anderen Formen von Nitrosyl-Komplexen. Aus der Nitrosierung von Ras und zwar des Restes C118 resultiert ein "Apo" Zustand ohne jegliches Nukleotid in Gegenwart einer oxidierenden RNS oder RS Umgebung. Zellbasierte Assays haben gezeigt, dass Ruthenium-Nitrosylkomplexe das Fortschreiten von Krebs unter kontrollierten Bedingungen im Vergleich zu anderen Formen von

147 135 Nitrosylkomplexen spezifisch um ungefähr 60% hemmen. Abgesehen von ihrer Antikrebs- Aktivität wird die Rolle der Nitrosierung unter dem Gesichtspunkt einer GTPase-Aktivität untersucht. Unter kontrollierten Bedingungen ist die intrinsische GTPase-Aktivität in Gegenwart der RNS und RS formen von Ruthenium-Nitrosylkomplexen verzögert. Die Wechselwirkung zwischen den Effektor proteinen und der GTPase werden durch RNS und RS nicht beeinflusst. Weiter vom MPI Dortmund zur verfügung gestelltes semisynthetisches Ras protein zeigte keine hydrolyseaktivität in bezug auf gebundenes GTP.

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156 144 8 Appendix 8.1 Mass spectrum of Zinc Cyclen Sulfate Zinc Cyclen, Sulfate m/z 8.2 UV-Vis characterization of the nitroso and peroxo metal complexes after UV irradiation (duration of irradiation is shown in the box) Absorbance Titanium Peroxo Citrate 5 Min 10 Min 15 Min 20 Min 25 Min 30 Min 35 Min Before Wavelength, nm

157 145 RuA UV characterization 2 Ru Nitrosyl Complex Before 5 Min 10 Min 15 Min Absorbance Wavewlength, nm 8.3.a Photolysis Difference Spectrum of Titanium Peroxo Citrate.

158 b Photolysis Difference Spectra of Ruthenium Nitroso Complex, unlabeled nitric oxide is shown in blue, labeled nitric oxide is shown in red. 8.4 PDB structures and distances between different phosphates: X is the distance between γ P and β P, while Y is the distance between β P and α-p. Distances are in Å. - P - - P P - γ-p β-p α-p

159 147 PDB X Y Type 1XCM 1X1S 1IAQ 821P 721P 621P 1AGP 421P 221P 5P21 2Q31 1A2B 121P 6Q21 1JAH 2RAP 3RAP 1C1Y 1QRA 521P 2UZI 1CRP 1CRQ 4Q21 2Q21 1CRR 1EFM 1WQ GppNHp GppNHp GppNHp GppNHp GppNHp GppNHp GppNHp GppNHp GppNHp GppNHp GppNHp GTPγS GppCH 2 p GppCH 2 p GppCH 2 p GTP GTP GTP GTP GTP GTP GDP GDP GDP GDP GDP GDP GDP:AlFx

160 SDS PAGE of the semisynthetic Ras protein a b 8.6 Amplitude Spectra of AcCN and DMS interaction with Ras protein Rate k 1 transistion

161 149 Rate k 2 transistion 8.7 Electrostatic Potential charges of T1, T2, & T3

162 Calculated IR spectra of T1, T2, T3 T1 T2 T3

163 Mass Spectrum of truncated Ruthenium bioconjugates of NF1 peptide as shown in the figure 3.24.c, masses correspond to C(821.07), D(780.04) and peptide of B without metal at Intensity Mass 8.10 Hydrolysis difference Spectra of Ras and Q61A in the presence of 2X ruthenium NF1 peptide bioconjugate Control Ras WT protein (Black), Ras WT with 2X Ruthenium NF1 Peptide bioconjugate (Red), Q61A with 2X Ruthenium NF1 Peptide bioconjugate (Green).