Eva de Alba 1 From the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain

Size: px
Start display at page:

Download "Eva de Alba 1 From the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain"

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 47, pp , November 20, by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Structure and Interdomain Dynamics of Apoptosis-associated Speck-like Protein Containing a CARD (ASC) * S Received for publication, May 25, 2009, and in revised form, August 20, 2009 Published, JBC Papers in Press, September 15, 2009, DOI /jbc.M Eva de Alba 1 From the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain The human protein ASC is a key mediator in apoptosis and inflammation. Through its two death domains (pyrin and CARD) ASC interacts with cell death executioners, acts as an essential adapter for inflammasome integrity, and oligomerizes into functional supramolecular assemblies. However, these functions are not understood at the structural-dynamic level. This study reports the solution structure and interdomain dynamics of full-length ASC. The pyrin and CARD domains are structurally independent six-helix bundle motifs connected by a 23-residue linker. The CARD structure reveals two distinctive characteristics; helix 1 is not fragmented as in all other known CARDs, and its electrostatic surface shows a uniform distribution of positive and negative charges, whereas these are commonly separated into two areas in other death domains. The linker adopts residual structure resulting in a back-to-back orientation of the domains, which avoids steric interference of each domain with the binding site of the other. NMR relaxation experiments show that the linker is flexible despite the residual structure. This flexibility could help expand the relative volume occupied by each domain, thus increasing the capture radius for effectors. Based on the ASC structure, a tentative model is proposed to illustrate how ASC oligomerizes via CARD and pyrin homophilic interactions. Moreover, ASC oligomers have been analyzed by atomic force microscopy, showing a predominant species of disk-like particles of 12-nm diameter and 1-nm height. Taken together, these results provide structural insight into the behavior of ASC as an adapter molecule. Apoptosis and inflammation are biologically related processes that depend on multiple protein-protein binding events leading to the formation of apoptotic and inflammatory complexes. These interactions are to a large extent mediated by members of the death domain superfamily, which comprises * This work was supported by the European Commission Marie Curie International Reintegration Grant IRG (to E. d. A.), by the Spanish Ministerio de Ciencia e Innovación through the Programa Ramón y Cajal (to E. d. A.), by Plan Nacional I D I Grant BFU (to E. d. A.), and by Comunidad de Madrid and Agencia CSIC through the IV PRICIT CCG08- CSIC/SAL-3777 (to E. d. A.). S The on-line version of this article (available at contains supplemental Tables S1 and S2 and Figs. S1 S4. The atomic coordinates (code 2KN6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ ( 1 To whom correspondence should be addressed. Fax: ; dealbae@cib.csic.es. four family subclasses (1): i.e. death domains (DD), 2 death effector domains (DED), caspase recruitment domains (CARD), and pyrin domains (PYD). Most members of this superfamily are composed of multiple domains, typically from two to six, which mediate homotypic interactions within each domain subfamily (1). In particular, DD/DD, DED/DED and CARD/CARD interactions have been characterized structurally (1), whereas the PYD/PYD binding mode is currently unknown. The human protein ASC (2, 3) is a member of the death domain superfamily bearing two death domains (N-terminal PYD, C-terminal CARD). ASC functions as an adapter molecule in both apoptosis and inflammation by interacting with Bax (4, 5) and caspases (6, 7) during apoptosis and by regulating the caspase-1-dependent inflammatory form of cell death named pyroptosis (8). ASC involvement in the cell death machinery seems to be connected to human diseases such as cancer. In fact, ASC gene transcription is impeded by aberrant DNA methylation in numerous types of human cancer (breast, ovarian, brain, and prostate) (9). Additionally, ASC acts as an integral adapter in the assembly of the inflammasome, a multiprotein complex necessary to activate caspase-1 leading to the processing and secretion of proinflammatory cytokines (10 12). The inflammasome comprises the NOD/NACHT-LRR proteins, ASC, and caspases 1 and 5 (13). NOD/NACHT-LRR proteins (14) recognize pathogenassociated molecular patterns and lead to a cascade of interactions responsible for caspase-1 activation. This molecular cascade regulates the processing of interleukin-1 family members. The several types of inflammasomes identified to date differ in the NOD/NACHT-LRR protein (Ipaf, NALP1, NALP2, cryopyrin/nalp3, and pyrin) and the cellular mechanism followed to activate caspase-1. The NALP2-, cryopyrin/nalp3-, and pyrin-dependent inflammasomes do not associate directly with caspase-1 and require ASC as an adapter (15 19). This assembly is mediated by homophilic interactions between the PYD and CARD of ASC with the PYD-containing NOD/NACHT-LRR and the CARD of procaspase-1 (13, 15, 20, 21). In addition, ASC oligomerizes into supramolecular and functional complexes. For instance, inflammatory stimuli in macrophages induce the formation of a large ASC complex named pyroptosome, which is a potent caspase-1 activator responsible 2 The abbreviations used are: DD, death domain; DED, death effector domain; CARD, caspase recruitment domain; PYD, pyrin domain; ASC, apoptosisassociated speck-like protein containing a CARD; NOE, nuclear Overhauser effect; AFM, atomic force microscopy; HSQC, heteronuclear single quantum correlation; 3D, three-dimensional; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 NUMBER 47 NOVEMBER 20, 2009

2 for pyroptosis (8). This ASC assembly has been observed in vitro as well (22). During apoptosis, ASC also oligomerizes into structures (2, 6) that serve as the scaffold for supramolecular platforms involved in caspase activation (13, 18). The multiple functions of ASC have granted it the nickname of molecular glue (10). ASC is an attractive candidate in the death domain superfamily for structural studies because it performs critical roles in apoptosis and inflammation with the simplest molecular architecture consisting of two domains. The structural and dynamic characterization of full-length ASC could help to improve our current understanding of the role played by interdomain conformational dynamics and the interplay between domains in the function of death domain proteins. What is known structurally on this subject emerges from the reported 3D structures of only two proteins with more that one death domain: i.e. FADD (composed of a DD and a DED) (23) and MC159 (a tandem of two DEDs) (24). The structure of FADD shows a tail-to-tail orientation of its two domains that results from the short (six-amino acid) linker connecting them and few contacts between both domains located in the vicinity of the linker. In contrast, the two DEDs of MC159 form a binding interface, as expected from the propensity of death domains to form homotypic interactions within members of the same subfamily (1). To date, the 3D structure of the pyrin domain of ASC is known (25). On the basis of this structure it has been suggested that the PYD/PYD interaction is analogous to the CARD/CARD binding mode (26, 27). However, the absence of the CARD in this structure precludes investigating the interplay between domains in ASC function. This study reports the high resolution NMR structure of fulllength ASC together with the analysis of its interdomain dynamics using NMR relaxation techniques. The results show that the PYD and CARD of ASC are structurally independent and connected by a flexible linker. The linker displays some local structure that restrains interdomain dynamics, leading to a back-to-back orientation of the two domains that facilitates binding to multiple partners. The interdomain flexibility of ASC could operate in a fly-casting fashion (28) to increase its ability to capture binding partners. By combining the ASC structure with the single known 3D structure of a CARD/CARD complex (27), a model for ASC dimerization is proposed. The model illustrates that the PYD and CARD of ASC are confined in space without obstructing the binding of each domain to their respective partners and suggests a possible way to oligomerize into larger assemblies. To complement the high resolution structural and dynamics studies by NMR, ASC oligomers have been analyzed by atomic force microscopy showing that ASC oligomerizes into 12-nm-diameter and 1-nm-height disk-like particles. The results reported herein provide structural and dynamic insights into how ASC can operate as molecular glue through protein-protein interactions mediated by its two death domains. EXPERIMENTAL PROCEDURES Protein Cloning, Expression, and Purification Cloning, expression, and purification of human ASC has been reported elsewhere (29). NMR Spectroscopy for Structural Studies NMR samples were prepared at 0.2 mm ASC, 5 mm d 15 -Tris(2-carboxyethyl)phosphine, 0.1 mm NaN 3, ph 3.8, 5% D 2 O/H 2 O, and 100% D 2 O. NMR experiments were acquired at 298 K in a Bruker Avance 600 MHz spectrometer equipped with a triple-resonance cryogenic probe. Sequence backbone assignments were obtained from the following experiments: [ 1 H, 15 N]-HSQC, 3D HNCO, 3D HNCACB, and 3D CBCA(CO)NH. Side-chain assignments were obtained from 3D HBHA(CO)NH, 3D (H)CC(CO)NH-TOCSY, and 3D H(CCCO)NH-TOCSY. NOE data were obtained from 3D 15 N-[ 1 H, 1 H]-NOESY (90-ms mixing time) and four-dimensional [ 1 H- 13 C, 1 H- 13 C]-NOESY (90-ms mixing time). Information on NMR experiments for protein structure determination can be found elsewhere (30, 31). All experiments were processed with NMRPipe (32) and analyzed with PIPP (33). Spectra resulting from some of these experiments are shown in Fig. 1 and supplemental Fig. S1. Several attempts were made to obtain residual dipolar couplings, including the use of bicelle (34) and bacteriophage (35, 36) alignment systems. ASC appears to disrupt bicelle formation resulting in the absence of protein alignment. In the presence of bacteriophages, the NMR spectrum mostly shows signals corresponding to the flexible linker, therefore, precluding the measurement of sufficient data to include in the structure calculation protocol. Structure Calculation Peak intensities from NOESY experiments were translated into a continuous distribution of interproton distances. Distances involving methyl groups, aromatic ring protons, and non-stereospecifically assigned methylene protons were represented as a summation averaging, ( r 6 ) 1/6 (37). Errors of 40 and 30% of the distances were applied to obtain lower and upper distance limits. 78 hydrogen bond distance restraints (r NH-O Å, r N-O Å) were defined according to the experimentally determined secondary structure of the protein. The TALOS program (38) was used to obtain 311 and restraints for those residues with statistically significant predictions. Structures were calculated with the program X-PLOR-NIH (39). The starting structure was heated to 3000 K and cooled in 30,000 steps of ps during simulated annealing. The final ensemble of 20 NMR structures was selected based on lowest energy and no restraint-violation criteria. The 20 lowest energy conformers have no distance restraint violations and no dihedral angle violations greater than 0.35 Å and 4.5, respectively. Structure quality was assessed with PROCHECK-NMR (40) and MolProbity (41). Structures were analyzed with MOLMOL (42). Coordinates were deposited in the Protein Data Bank with accession code 2KN6. Backbone 15 N Relaxation Measurements Relaxation experiments were performed at 298 K in a Bruker Avance spectrometer operating at 600 MHz. The 15 NT 1,T 1, and { 1 H}- 15 N NOE data were obtained with specific NMR pulse sequences (43, 44). The recycle delay to measure 15 NT 1 and T 1 was 1 s, whereas { 1 H}- 15 N NOE experiments used 3.2s. All experiments were acquired in an interleaved manner to minimize the effects caused by spectrometer drift. The relaxation delays of T 1 experiments were the following: 12, 36, 100, 244, 484, 964, 1284, and 1604 ms. T 1 experiments used a 15 N continuous spin-lock field NOVEMBER 20, 2009 VOLUME 284 NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 32933

3 of 2.5 khz. T 1, instead of T 2 relaxation times were acquired because resonance offset effects are significant in T 2 experiments, whereas they can be corrected in a straightforward manner for T 1 data using the equation (45), 1/T 1 cos 2 /T 2 sin 2 /T 1 (Eq. 1) where tan 1 ( N / N B 1 ), N is the resonance offset, and N B 1 is the strength of the spin-lock field. T 2 values can be obtained from Equation 1, as T 1,T 1, and are known. Relaxation times were calculated by fitting peak-intensity dependence with the experiment relaxation times to an exponential function given by I(t) I 0 e[( 1/T)t] (T T 1,T 1 ). T 1 and T 1 values are averages of two separate measurements. The { 1 H}- 15 N NOE values were calculated from the ratio of peak intensities obtained from experiments performed with and without 1 H presaturation. The 1 H frequency was shifted offresonance in the unsaturated experiments. The pulse train used for 1 H presaturation utilized 162 pulses separated by 50-ms delays and was applied for a total of 2.2s. The recycle time is reasonably long; however, NOE values were corrected for incomplete 1 H magnetization recovery as previously described (44). Apparent rotational correlation times were obtained assuming full anisotropy, as described elsewhere (46), from relaxation data of residues that do not undergo slow conformational averaging and show { 1 H}- 15 N NOE values larger than 0.65 (43). The parameters of the rotational diffusion tensor are shown in supplemental Table S1. Atomic Force Microscopy Imaging Mica surfaces were covered with 3 l of protein solution at either ph 3.8 or 7.0 and incubated for 30 s. The surface was subsequently rinsed with the respective buffers at ph 3.8 and 7.0 and dried. Tapping mode imaging in air was conducted with a Multimode Atomic Force Microscope (Veeco Instruments, Santa Barbara, CA) using a Nanoscope IIIa controller and a J scanner. Veeco nanoprobe tips TESP7 with a resonance frequency of 320 khz and a spring constant k 42 newtons/m were used. Scan rates were set at 1Hz. Molecular Modeling The solution structure of ASC was used as the monomer template to build the model for the ASC dimer by superimposing the CARD of ASC to Apaf-1-CARD and caspase-9-card complex (27). The model was created with the program MOLMOL (42). RESULTS ASC Propensity to Oligomerize by NMR and AFM ASC selfassociates in vivo during apoptosis and inflammation (2) and is capable of forming functional supramolecular assemblies in vitro (22). The oligomerization of ASC poses significant challenges for NMR structural studies regarding protein solubility and particle size. It is, therefore, critical to find conditions to minimize oligomerization at the relatively high concentrations used in protein NMR ( 1mM). ASC solubility is very low at neutral ph. The soluble fraction of ASC at ph 7 cannot be detected with Coomassie Blue staining in polyacrylamide gel electrophoresis (detection limit ng) and results in few observable signals with intensity FIGURE 1. ASC is properly folded at ph 3.8 and oligomerizes at concentration 1mM. A, shown is a [ 1 H, 15 N]-HSQC spectrum of 13 C, 15 N-labeled ASC at ph 3.8. Residue numbers are assigned to the corresponding signals. Assignment of the central region of the spectrum is not shown for clarity. sc denotes side chain. B, shown is the 1 H amide region in one-dimensional projections of [ 1 H, 15 N]-HSQC spectra of 0.7 mm 13 C, 15 N-labeled ASC at ph 3.8. The black, green, and red lines correspond to spectra acquired 1h, 2 h, and 5 days after sample preparation, respectively. slightly above the noise level in a spectrum acquired using fast NMR acquisition techniques (47) (supplemental Fig. S2). In contrast, under acidic conditions (ph 4) ASC solubility improves and can readily be detected by NMR. The dispersion of NMR signals in the [ 1 H, 15 N]-HSQC spectrum of ASC (Fig. 1A and supplemental Fig. S2) indicates that the protein is properly folded at this ph. However, NMR signal intensity decreases over time with no concomitant changes in the chemical shifts (Fig. 1B). This result suggests that ASC forms oligomers of considerable size that tumble too slowly to be observed in the NMR spectrum. The NMR signal intensity of 0.7 mm ASC decays to 80% after 3 h of sample preparation and to 30% within the first 5 days (Fig. 1B). In contrast, at 0.2 mm protein concentration, signal intensity decays to 98% after 3 h of sample preparation and to 80% within the first 24 h. NMR signal intensity continues decaying to 75% of the original signal, reaching a plateau after 3 days of sample preparation. Therefore, by decreasing the protein concentration to 0.2 mm, the effect of ASC oligomerization in NMR signal intensity is significantly reduced, whereas it is still possible to acquire NMR triple-resonance spectra with a signal enhancing cryogenic probe. Because the capability of ASC to oligomerize is basic to its function, oligomerization at neutral and acidic ph was investi JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 NUMBER 47 NOVEMBER 20, 2009

4 FIGURE 2. Tapping mode AFM images of ASC oligomers. Left, shown are amplitude images of ASC oligomers (circled)atph7.0(a) and ph 3.8 (B). Right, shown are sections of height images of ASC oligomers circled in blue (A) and blue and red (B). The total section in A flanks two particles simultaneously and is, therefore, two times the length (70 nm) of the section in (B) (35 nm), which flanks each particle individually. TABLE 1 Rotational correlation times ( c ) System No. of structured residues a c (ns) b c (ns) Full-length ASC ASC-PYD ASC-CARD ASC c 6.3 Calmodulin N-terminal domain c 4.1 Calmodulin C-terminal domain c 3.7 a Experimental values from magnetic relaxation. b Theoretical values obtained with a spherical model (49) using the number of residues specified and the temperature corresponding to each case: ASC (298 K), ASC2 (298 K), and calmodulin (308 K). c Reported experimental values: ASC2 (56), calmodulin (43). gated by scanning atomic force microscopy. AFM images show that ASC forms oligomers of similar size and shape at both ph values (Fig. 2, A and B). The predominant species appears like disks of 1-nm height and 12-nm diameter (see the section images in Fig. 2, A and B). Taken together, the NMR and AFM data indicate that ASC is able to oligomerize in vitro into particles of similar structural features under both ph conditions. Based on these results, it is reasonable to assume that the structure of ASC is not perturbed at acidic ph. Under acidic conditions, the oligomerization reaction likely favors the monomeric Structure and Interdomain Dynamics of ASC form, increasing in turn the solubility of ASC and, thus, leading to a larger fraction of monomers observable by NMR. To investigate whether the possible oligomerization of ASC at 0.2 mm interferes with structural and dynamics studies, NMR relaxation measurements were performed. Backbone amide ( 15 N) magnetic relaxation experiments provide rotational correlation times ( c ), which directly depend on the molecular size and shape (46, 48). In the presence of aggregation, measured c values should be larger than theoretical values derived from the molecular size (49). Differences between the two can also originate from the low sphericity of the protein and from dynamic processes associated, for example, to interdomain motion. The former case generally results in experimental c values larger than the prediction. The experimental c value of ASC is small for its size ( 22 kda) (Table 1), indicating that the fraction of ASC molecules observable by NMR tumbles as monomers. The discrepancy with the theoretical value (Table 1) could, therefore, be due to interdomain dynamics in ASC (see below). In addition, amide 15 N transverse relaxation times (T 2 ) depend on the protein rotational correlation time but are reduced in the presence of aggregation (50). For ASC, the average T 2 value of the two domains ( ms) agrees with the measured correlation time. These results indicate that ASC oligomerization does not have significant effects under the conditions used for the following NMR structural and dynamics studies. High Resolution Structure of Full-length Human ASC The three-dimensional structure of ASC was determined with 3046 NOE-derived distances and 311 dihedral and 78 hydrogen bond restraints. The 20 lowest energy conformers of ASC do not show distance or angle restraint violations greater than 0.35 Å and 4.5, respectively (Table 2). The ensemble of structures does not show significant deviations from covalent geometry and is well defined by the NMR data, resulting in low atomic coordinate precision for the backbone and all heavy atoms (Table 2). Structural validation data of the ASC structure obtained with MolProbity (41) in comparison to average values calculated for all NMR PYD and CARD structures deposited in the Protein Data Bank indicate that the structure of ASC is of comparable quality (Table 3). The equivalent resolution provided by PROCHECK-NMR (40) of the ASC structure compared with x-ray structures is between 1 and 1.8 Å (supplemental Fig. S3). NOVEMBER 20, 2009 VOLUME 284 NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 32935

5 TABLE 2 Structural statistics of human ASC Statistics were calculated for the 20 conformers with the lowest overall energies and no NOE or dihedral angle restraint violations greater than 0.35 Å and 4.5, respectively. Root mean square deviations 20 lowest energy conformers Lowest energy conformer Restraints Distances, Å (3046) Intraresidue (1547) Sequential i j 1 (497) Short range i j 5 (511) Long range i j 5 (491) Hydrogen bonds, Å (78) Dihedrals (, ) (311) Deviations from ideal covalent geometry Bonds, Å Angles Impropers Structure quality Lennard-Jones potential energy (kcal mol 1 ) a Ramachandran (PROCHECK-NMR (40)) 88% (residues in most favored regions) 1.4% (residues in disallowed regions) 88% (residues in most favored regions) 1.4% (residues in disallowed regions) Coordinate precision, Å Residues 3 88 Residues Backbone heavy atoms All heavy atoms a The Lennard-Jones van der Waals energy was calculated with the CHARMM PARAM19/20 parameters and was not included in structure calculation. TABLE 3 MolProbity (41) structure validation for the representative structure of ASC and average values for the reported PYD and CARD NMR structures ASC Average value a All-atom contacts, Clashscore for all atoms b,c MolProbity Score b Ramachandran outliers 4.7% 3.0% Ramachandran favored 90.2% 85.8% Rotamer outliers 9.5% 14.5% C deviations 0.25 Å Residues with bad bonds 0% 1.0% Residues with bad angles 0% 0.3% a Average values were calculated using the representative model of the reported CARD and PYD NMR structures: isolated PYD of ASC (25), NALP1-PYD (55), ASC2 (56), NALP10-PYD (PDB entry 2DO9), PYD of myeloid cell nuclear differentiation antigen (PDB entry 2DBG), Apaf-1-CARD (26, 65), ICEBERG (66), RAIDD-CARD (67), NOD1-CARD (Ref. 68, and PDB entry 2DBD). b 100 is the best structure quality score. c Clashscore is the number of serious steric overlaps ( 0.4 Å) per 1000 atoms. The NMR structure of full-length ASC shows two six-helix bundle domains (PYD and CARD) connected by a 23-residuelong linker (Fig. 3A). No interdomain NMR-derived contacts (NOEs) were observed, and neither domain shows NOEs with the linker; therefore, the PYD and CARD do not interact with each other. This result agrees with the previously observed propensity of death domains to participate in homotypic interactions within each subfamily (1) and the description of ASC as an adapter protein with two homophilic protein-protein interacting domains (51). Although long, the linker of ASC (residues ) adopts some residual structure as evidenced by the presence of short-range NOEs. In addition, the 13 C chemical shifts (29) deviate from random coil values. NOE data involving residues (supplemental Table S2) and some positive 13 C secondary shifts in this region (Fig. 4A) suggest that it adopts residual turn-type conformations. In contrast, 13 C chemical shift deviations are almost consistently negative from residues 95 to 112 (Fig. 4A), pointing to the formation of low populated extended structures (52). These results are supported by the empirical program TALOS (38), which using a combination of ASC chemical shifts of several nuclei ( 15 N amide, 13 C, 13 C, 13 C, 1 H ) as input data, predicts most linker residues to populate extended structures (Fig. 4B). It is noteworthy that only five residues fall outside the extended structure region of the Ramachandran plot. Three of them (Gln-91, Gly-92, and Gly-94) belong to the fragment that, based on NOE data, is likely adopting turn or short-helix conformations. The other two residues (Gly-99 and Gly-111) are comprised in the fragment and fall in the left-handed helix region characteristic of Gly. The propensity of the linker to adopt extended structure is not surprising on the basis of its amino acid composition. Residues such as Ser, Ala, Gly, and Pro, present in the linker of ASC, are known to bias the polypeptide chain toward such conformations (53 54). In particular, two consecutive Pro residues (Pro-103 and Pro-104 in the linker of ASC) favor the polyproline II or collagen conformation (54), which is a common residual extended structure found in protein loops and linkers connecting domains in modular proteins (53). In contrast to the structure of full-length ASC reported here, in the solution structure of the protein FADD, few interdomain NMR contacts have been observed between the DD and the DED (23). These interactions are not located in the consensus binding sites of each domain and are spatially close to the short linker (6 amino acids) connecting them. The linker length and flexibility could, thus, emerge as important factors in the structure and dynamics of the death domain superfamily. The PYD of full-length ASC shares some general characteristics with other known PYD structures, including the long loop between helices 2 and 3 (Figs. 3A and 4), a feature of PYDs absent in other death domains. However, it is worth noting several differences with the isolated PYDs of NALP1 (55) and NALP10 (PDB entry 2DO9). These show a disordered loop in place of helix 3 in the PYD of ASC (Fig. 3B). Helices 1 and 6 are also shorter in the PYD of NALP1 (Fig. 3B). These structural differences result in high root mean square deviation values (9.7 Å for NALP1-PYD and 7.3 Å for NALP10-PYD) and might be related to their differences in biological function. In fact, JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 NUMBER 47 NOVEMBER 20, 2009

6 FIGURE 4. A, ASC 13 C chemical shifts deviations from random coil values (64) versus residue number are shown. The helices and interdomain linker are indicated. B, a Ramachandran representation of and values predicted by TALOS (38) for residues in the linker of ASC is shown. Red circles highlight residues in the fragment not predicted to populate extended conformations. Most residues in the fragment are clustered in the extended structure region with the exception of Gly-99 and Gly-111. Bars indicate uncertainties in the predicted values. FIGURE 3. Solution structure of full-length human ASC and comparison to representative PYD and CARD known structures. A, shown is a ribbon representation of the solution structure of full-length human ASC. Helices of the CARD are colored in dark green (front) and light green (back). Helices of the PYD are colored in red (front) and orange (back). B, left panel, shown is the superposition of the structures of full-length ASC PYD (red) and NALP1- PYD (blue) (55). Right panel, shown are full-length ASC PYD (red) and NALP10- PYD (orange) (PDB entry 2DO9). C, shown is the superposition of the structures of full-length ASC CARD (green) and Apaf-1-CARD (navy) (26). Helices are shown as cylinders in B and C. D, electrostatic surface of full-length ASC CARD and Apaf-1-CARD (only the negatively charged area is shown in Apaf-1- CARD). Protein orientation is equivalent. NALPs contain additional domains and leucine-rich motifs, suggesting different roles in inflammasome formation. In contrast, the PYD-only protein ASC2 (56), which is thought to modulate ASC-mediated autoimmune response through PYD/ PYD interactions (57), is significantly similar to the PYD of ASC at the structural level (root mean square deviation 1.36 Å) and also displays the helix 3. Not surprisingly, the PYDs of both proteins share a high degree of sequence identity (64%). The PYD of full-length ASC and the isolated PYD (25) are structurally very similar as well (root mean square deviation 1.37 Å). This result indicates that the presence of the CARD does not perturb the structure of the PYD in ASC and agrees with the absence of interdomain contacts. Full-length ASC CARD shows structural peculiarities compared with other known CARDs. In all previously reported CARD structures, helix 1 is bent or broken into two smaller NOVEMBER 20, 2009 VOLUME 284 NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 32937

7 FIGURE 5. Backbone 15 N amide NMR relaxation data of full-length ASC. A, amide { 1 H}- 15 N NOE values versus residue number are shown. Arrows indicate PYD and CARD length. B, 15 N relaxation time ratios (T 1 /T 2 ) versus residue number are shown. Straight lines indicate average T 1 /T 2 values of each domain. helices, named H1a and H1b. The hinge connecting these two fragments is involved in protein-protein interactions according to structural studies on the complex between Apaf-1-CARD and caspase-9-card (26, 27). Fragment H1a is missing in the CARD of ASC. Helix 1 spans residues Gln-117 to Val-126 and is preceded by a relatively ordered turn (His-113 Asp-116) (Fig. 3A). A comparison of the CARD structure of full-length ASC with the solution structure of Apaf-1-CARD (26), which is one example with the two H1a and H1b fragments, is shown in Fig. 3C. The H1a fragment displayed by Apaf-1-CARD corresponds to residues in ASC. The region is significantly flexible according to NMR relaxation data (see below), thus confirming the absence of H1a in ASC. The lack of H1a in the CARD of ASC might be related to its plasticity in proteinprotein interactions that could facilitate participation in apoptotic and inflammatory events. In addition, large deviations in the orientation of helices 2 and 3 are also observed (Fig. 3C), which can result from the propagation of structural changes in the binding surface involving the connection between H1a and H1b. The electrostatic surface of full-length ASC CARD is significantly different from Apaf-1 CARD (Fig. 3D). ASC shows positively and negatively charged areas evenly spread throughout the surface, whereas Apaf-1-CARD shows two extensive oppositely charged patches (Fig. 3D). Within a similar fold, CARDs show structural differences pertaining to helix length, orientation (1), and electrostatic surface (examples are illustrated in supplemental Fig. S4), which might serve as a finetuned mechanism to tightly control the binding specificity observed in protein-protein interactions mediated by these domains. Interdomain Dynamics in ASC NMR relaxation of backbone amide ( 15 N) measured as heteronuclear Overhauser values ({ 1 H}- 15 N NOE) as well as longitudinal (T 1 ) and transverse (T 2 ) relaxation times are affected by N-H bond dynamics and the molecule s rotational diffusion (58, 59). Residues adopting FIGURE 6. Superposition of the 10 lowest energy conformers of fulllength human ASC. A, individual superposition of the PYD is shown. Helices are numbered and colored in red (front helices) and orange (back helices). The linker and CARD are colored in yellow and green, respectively. B, individual superposition of the CARD is shown. Helices are numbered and colored in dark green (front helices) and light green (back helices). The linker and PYD are colored in yellow and orange, respectively. regular secondary structure show heteronuclear NOE values close to the theoretical maximum ( 0.83 at a spectrometer frequency of 600 MHz), whereas values lower than 0.65 are symptomatic of internal dynamics (43, 60). The average heteronuclear NOE values for the PYD and CARD regions of ASC are high and similar ( and , respectively) as expected for two rigid structures (Fig. 5A). In contrast, heteronuclear NOE values decrease from the PYD C terminus and the CARD N terminus toward the linker center (Fig. 5A). These results indicate that the linker undergoes local motions on a fast time scale compared with molecular tumbling. These motions become increasingly restricted toward the connections to the DDs. Thus, the heteronuclear NOE data indicate that ASC comprises two well ordered rigid domains connected by a flexible linker JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 NUMBER 47 NOVEMBER 20, 2009

8 FIGURE 7. Molecular model of the ASC dimer and representation of the interacting region of the PYD in the electrostatic surface and as a ribbon diagram. A, left, shown is a model of the ASC dimer. The 10 lowest energy conformers of ASC CARD are superimposed and colored in red and dark blue. PYDs are colored in yellow and light blue. Right, CARDs in the dimer model showing the relative orientation of the central axes (green cylinders) that form an angle of The central axes and angle were calculated with the program MOLMOL (42) using the C atoms of the fragment comprising helices 4 and 5, which is in the center of the CARD structure. B, left, shown is the electrostatic surface of human ASC indicating the PYD. Negatively and positively charged surfaces are colored in red and blue, respectively. Right, a ribbon diagram of ASC PYD inside its volume is shown. The negatively and positively charged helices suggested to interact are highlighted in red and blue. The remaining helices are orange. The 10 lowest energy conformers of the CARD are shown (green) to indicate their accessible space. The linker is colored in yellow. The orientation of the PYD in the left and right figures is the same to show the electrostatic surface of the interacting helices. The next step is to analyze the dynamic behavior of each domain relative to the other. Two extreme models for ASC interdomain dynamics can be envisioned; in the first, both domains tumble as a single rigid body, and in the second model, each domain is dynamically independent. Backbone amide 15 N T 1 /T 2 ratios are particularly useful in this type of analysis, as they are similar among the domains when they tumble as a whole and different otherwise (43). The 15 NT 1 /T 2 ratios of the PYD and CARD of ASC are noticeably different, indicating that they reorient at different rates (Fig. 5B). In addition, NMR relaxation-derived c values of ASC individual domains are significantly larger than the predicted values (Table 1). Theoretical and NMR c values of globular proteins are generally in very good agreement. As an example, Table 1 shows the theoretical and experimental c values of the PYD-only protein ASC2 (56) of similar size and structure to ASC individual domains. The NMR c values of both PYD and CARD in ASC are also larger than the experimental c of ASC2, indicating that the former do not tumble independently. These results suggest that the PYD and CARD in ASC are in between the two extreme models, Structure and Interdomain Dynamics of ASC showing some interdomain flexibility and simultaneously sensing each domain the drag of the other. This behavior is structurally illustrated by superimposing each individual domain of the NMR conformational ensemble (Fig. 6). The residual structural preferences of the linker result in a defined spatial interdomain organization that determines the orientation for binding of one domain relative to the other. Within this topological arrangement the flexibility of the linker increases the accessible space sampled by each domain, improving therefore, the chances to find interacting partners relative to proteins with two structurally fixed domains. Interdomain motions caused by linker flexibility have been related to protein function. A classical example is the NMR study on the dynamic behavior of the two-domain protein calmodulin (43). This study shows that the long interdomain linker is highly mobile, supporting its role in calmodulin versatility to bind to multiple partners. Moreover, the linker flexibility is proposed to allow both protein halves to simultaneously interact with the target and to adopt the different domain orientations required in the formation of each complex. The binding requirements of ASC and calmodulin are analogous in that both proteins need to interact with different partners. Therefore, the flexibility of the linker could play a similar role in the interdomain dynamics of both proteins. Nevertheless, an important difference in the behavior of ASC could be that each domain binds at least one different target to form a particular complex. Like ASC, calmodulin domains show NMR relaxation-derived c values that are significantly larger than the prediction (Table 1) albeit smaller than the correlation time expected for a globular protein of similar size (43). Interdomain motions in calmodulin have been further studied, resulting in the determination of the motion time scale (61). Examples of proteins with domain dynamics fitting the first extreme scenario explained above have also been reported (62). In this case the domains orient together because short or rigid linkers connect them or because they participate in interdomain contacts. In the death domain superfamily, FADD serves as an example of two domains orienting as a whole (23). A Model for ASC Oligomerization ASC forms homo- and hetero-oligomeric assemblies that are tightly bound to its function. NMR data and AFM images reported here indicate that NOVEMBER 20, 2009 VOLUME 284 NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 32939

9 ASC is also capable of self-associating in vitro (Figs. 1 and 2). Based on this information and the propensity of death domains to form homotypic interactions within subfamilies, it is possible to build a model to illustrate how ASC could oligomerize. The model (Fig. 7A) uses the structure of full-length ASC as the monomer template together with the binding interface of Apaf- 1-CARD-caspase-9-CARD complex structure (27), which is currently the single three-dimensional CARD/CARD complex structure known. The structure of a PYD/PYD complex has not been determined up to date. The CARD/CARD interaction is asymmetric, involving helices 1 and 4 of one CARD and helices 2 and 3 of the other (Fig. 7A). The asymmetry leaves two free binding sites in the dimer: helices 1 and 4 of one monomer and helices 2 and 3 of the other. The free binding sites allow additional CARD/CARD interactions, naturally leading to oligomerization. PYD/PYD interactions are suggested to also involve helices 1 and 4 of one PYD and helices 2 and 3 of the other (25). The PYD/PYD interface would be asymmetric and, therefore, would leave free binding sites upon dimer formation. Thus, the PYDs could as well participate in the self-association of ASC through homophilic interactions. The formation of the CARD/CARD and PYD/ PYD interaction in the dimer model pre-establishes the relative binding orientation of the each domain to other partners (Fig. 7A). Moreover, in this model the pyrin domains are confined to a restrained space on top of the CARDs and do not obstruct the CARD/CARD interface (Fig. 7A). Interestingly, in the ASC structure the helices suggested to participate in the PYD/PYD interface are positioned as far as possible from the CARD and are, therefore, accessible for interactions with other partners (Fig. 7B). Thus, the CARD does not interfere with the PYD interface suggested to be involved in PYD/PYD interactions. The CARDs central axes in the dimer model are positioned at an angle (Fig. 7A) that could result in the formation of a ring upon further association through the free interacting surfaces. The value of this angle (53.6 ) is consistent with a ring composed of 6 7 monomers. Strikingly, electron microscopy data of the supramolecular apoptosome formed by Apaf-1 oligomerization show a 7-member CARD ring (63). In addition, the protein NALP1, which bears an N-terminal PYD and a C-terminal CARD flanking other domains, also forms a 7-fold symmetric ring as observed by electron microscopy (18). Both are rings of nm outer diameter. Interestingly, the disklike ASC oligomers (Fig. 2) of 12-nm diameter suggest that ASC could oligomerize into rings analogous to those formed by Apaf-1 and NALP1. However, ASC has been shown by confocal microscopy to also form rather large specks of 2 m in diameter (8) and filaments (2). These results together with the AFM data reported here suggest that the oligomerization of ASC could be a complex process. It is likely that the disk-like oligomers observed by AFM further aggregate into larger assemblies. DISCUSSION This study shows how the molecular architecture of ASC facilitates self-association and multiple binding to several proteins, which in turn could result in the assembly of supramolecular platforms. This role directly emerges from ASC interdomain orientation and dynamics. ASC interdomain topological organization facilitates binding by avoiding steric interference between the two domains and favors a specific protein binding orientation. In addition to spatial confinement, ASC shows interdomain flexibility, which is proposed to increase the search space of each domain independently, therefore, enhancing the probability to find interacting partners. The interdomain structural and dynamic properties of ASC are significantly different from FADD, which is the only other protein structure currently known with two death domains belonging to different subfamilies. The length of the linker (23 amino acids in ASC and 6 amino acids in FADD) could be partially responsible for the observed differences between the two proteins and, therefore, might emerge as an important factor in the operating mode of death domain proteins. The oligomerization of ASC is another distinctive characteristic related to its capability to form supramolecular assemblies. The absence of intramolecular PYD/CARD interactions in the structure of ASC agrees with all structural and biochemical data of death domains reporting their tendency to form homophilic interactions within each subfamily and suggests that ASC oligomerizes through homotypic interactions mediated by its CARD and PYD. The overall dimension and shape of these oligomers are reported here. Interestingly, ASC oligomers are disk-like particles of similar size to the Apaf-1 CARD and NALP1 rings. Taken together, the structural and dynamic features of ASC shed light into the function of this protein as an adapter molecule and its capability to form supramolecular complexes in apoptosis and inflammation. Further research in this area will help to establish whether other members of the death domain superfamily, with multiple protein-protein binding domains connected by relatively long linkers, share some of these structural features and behave similarly. Acknowledgments The gift of ASC cdna from Professor Gabriel Núñez is greatly appreciated. Dr. Nico Tjandra is acknowledged for software used in magnetic relaxation analysis. The suggestions of Professor Víctor Muñoz for manuscript preparation are also acknowledged. The assistance from Jörg Schönfelder in AFM experiments is greatly appreciated. REFERENCES 1. Park, H. H., Lo, Y. C., Lin, S. C., Wang, L., Yang, J. K., and Wu, H. (2007) Annu. Rev. Immunol. 25, Masumoto, J., Taniguchi, S., Ayukawa, K., Sarvotham, H., Kishino, T., Niikawa, N., Hidaka, E., Katsuyama, T., Higuchi, T., and Sagara, J. (1999) J. Biol. Chem. 274, Conway, K. E., McConnell, B. B., Bowring, C. E., Donald, C. D., Warren, S. T., and Vertino, P. M. (2000) Cancer Res. 60, Ohtsuka, T., Ryu, H., Minamishima, Y. A., Macip, S., Sagara, J., Nakayama, K. I., Aaronson, S. A., and Lee, S. W. (2004) Nat. Cell Biol. 6, Hasegawa, M., Kawase, K., Inohara, N., Imamura, R., Yeh, W. C., Kinoshita, T., and Suda, T. (2007) Oncogene 26, McConnell, B. B., and Vertino, P. M. (2000) Cancer Res. 60, Masumoto, J., Dowds, T. A., Schaner, P., Chen, F. F., Ogura, Y., Li, M., Zhu, L., Katsuyama, T., Sagara, J., Taniguchi, S., Gumucio, D. L., Núñez, G., and Inohara, N. (2003) Biochem. Biophys. Res. Commun. 303, Fernandes-Alnemri, T., Wu, J., Yu, J. W., Datta, P., Miller, B., Jankowski, W., Rosenberg, S., Zhang, J., and Alnemri, E. S. (2007) Cell Death Differ. 14, JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 NUMBER 47 NOVEMBER 20, 2009

10 9. McConnell, B. B., and Vertino, P. M. (2004) Apoptosis 9, Mariathasan, S. (2007) Microbes Infect. 9, Pétrilli, V., and Martinon, F. (2007) Joint Bone Spine 74, Taniguchi, S., and Sagara, J. (2007) Semin. Immunopathol. 29, Martinon, F., Burns, K., and Tschopp, J. (2002) Mol. Cell 10, Tschopp, J., Martinon, F., and Burns, K. (2003) Nat. Rev. Mol. Cell Biol. 4, Srinivasula, S. M., Poyet, J. L., Razmara, M., Datta, P., Zhang, Z., and Alnemri, E. S. (2002) J. Biol. Chem. 277, Stehlik, C., Lee, S. H., Dorfleutner, A., Stassinopoulos, A., Sagara, J., and Reed, J. C. (2003) J. Immunol. 171, Kanneganti, T. D., Ozören, N., Body-Malapel, M., Amer, A., Park, J. H., Franchi, L., Whitfield, J., Barchet, W., Colonna, M., Vandenabeele, P., Bertin, J., Coyle, A., Grant, E. P., Akira, S., and Núñez, G. (2006) Nature 440, Faustin, B., Lartigue, L., Bruey, J. M., Luciano, F., Sergienko, E., Bailly- Maitre, B., Volkmann, N., Hanein, D., Rouiller, I., and Reed, J. C. (2007) Mol. Cell 25, Eisenbarth, S. C., Colegio, O. R., O Connor, W., Sutterwala, F. S., and Flavell, R. A. (2008) Nature 453, Ogura, Y., Sutterwala, F. S., and Flavell, R. A. (2006) Cell 126, Mariathasan, S., Newton, K., Monack, D. M., Vucic, D., French, D. M., Lee, W. P., Roose-Girma, M., Erickson, S., and Dixit, V. M. (2004) Nature 430, Fernandes-Alnemri, T., and Alnemri, E. S. (2008) Methods Enzymol. 442, Carrington, P. E., Sandu, C., Wei, Y., Hill, J. M., Morisawa, G., Huang, T., Gavathiotis, E., Wei, Y., and Werner, M. H. (2006) Mol. Cell 22, Yang, J. K., Wang, L., Zheng, L., Wan, F., Ahmed, M., Lenardo, M. J., and Wu, H. (2005) Mol. Cell 20, Liepinsh, E., Barbals, R., Dahl, E., Sharipo, A., Staub, E., and Otting, G. (2003) J. Mol. Biol. 332, Zhou, P., Chou, J., Olea, R. S,, Yuan, J., and Wagner, G. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, Qin, H., Srinivasula, S. M., Wu, G., Fernandes-Alnemri, T., Alnemri, E. S., and Shi, Y. (1999) Nature 399, Shoemaker, B. A., Portman, J. J., and Wolynes, P. G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, de Alba, E. (2007) Biomol. NMR Assign. 1, Bax, A., and Grzesiek, S. (1993) Acc. Chem. Res. 26, Cavanagh, J., Fairbrother, W. J., Palmer, A. G., Rance, M., and Skelton, N. J. (2006) in Protein NMR Spectroscopy: Principles and Practice, Academic Press, Inc, San Diego, CA 32. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, Garret, D. S., Powers, R., Gronenborn, A., and Clore, G. M. (1991) J. Magn. Reson. 95, Tjandra, N., and Bax, A. (1997) Science 278, Hansen, M. R., Mueller, L., and Pardi, A. (1998) Nat. Struct. Biol. 5, Clore, G. M., Starich, M. R., and Gronenborn, A. M. (1998) J. Am. Chem. Soc. 120, Nilges, M. (1993) Proteins 17, Cornilescu, G., Delaglio, F., and Bax, A. (1999) J. Biomol. NMR 13, Schwieters, C. D., Kuszewski, J. J., Tjandra, N., and Clore, G. M. (2003) J. Magn. Reson. 160, Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., and Thornton, J. M. (1996) J. Biomol. NMR 8, Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J., Wang, X., Murray, L. W., Arendall, W. B., 3rd, Snoeyink, J., Richardson, J. S., and Richardson, D. C. (2007) Nucleic Acids Res. 35, W375 W Koradi, R., Billeter, M., and Wüthrich, K. (1996) J. Mol. Graph. 14, Barbato, G., Ikura, M., Kay, L. E., Pastor, R. W., and Bax, A. (1992) Biochemistry 31, Grzesiek, S., and Bax, A. (1993) J. Am. Chem. Soc. 115, Tjandra, N., Wingfield, P., Stahl, S., and Bax, A. (1996) J. Biomol. NMR 8, Tjandra, N., Feller, S. E., Pastor, R. W., and Bax, A. (1995) J. Am. Chem. Soc. 117, Schanda, P., and Brutscher, B. (2005) J. Am. Chem. Soc. 127, Brüschweiler, R., Liao, X., and Wright, P. E. (1995) Science 268, Daragan, V. A., and Mayo, K. H. (1997) Prog. NMR Spectrosc. 31, Wagner, G. (1993) J. Biomol. NMR 3, Moriya, M., Taniguchi, S., Wu, P., Liepinsh, E., Otting, G., and Sagara, J. (2005) Biochemistry 44, Spera, S., and Bax, A. (1991) J. Am. Chem. Soc. 113, Dyson, H. J., and Wright, P. E. (2005) Nat. Rev. Mol. Cell Biol. 6, George, R. A., and Heringa, J. (2002) Protein Eng. 15, Hiller, S., Kohl, A., Fiorito, F., Herrmann, T., Wider, G., Tschopp, J., Grütter, M. G., and Wüthrich, K. (2003) Structure 11, Natarajan, A., Ghose, R., and Hill, J. M. (2006) J. Biol. Chem. 281, Stehlik, C., Krajewska, M., Welsh, K., Krajewski, S., Godzik, A., and Reed, J. C. (2003) Biochem. J. 373, Palmer, A. G., Williams, J., and McDermott, A. (1996) J. Phys. Chem. 100, Ishima, R., and Torchia, D. A. (2000) Nat. Struct. Biol. 7, Kay, L. E., Torchia, D. A., and Bax, A. (1989) Biochemistry 28, Baber, J. L., Szabo, A., and Tjandra, N. (2001) J. Am. Chem. Soc. 123, Fushman, D., Varadan, R., Assfalg, M., and Walker, O. (2004) Prog. NMR Spectrosc. 44, Yu, X., Acehan, D., Ménétret, J. F., Booth, C. R., Ludtke, S. J., Riedl, S. J., Shi, Y., Wang, X., and Akey, C. W. (2005) Structure 13, Wishart, D. S., Bigam, C. G., Holm, A., Hodges, R. S., and Sykes, B. D. (1995) J. Biomol. NMR 5, Day, C. L., Dupont, C., Lackmann, M., Vaux, D. L., and Hinds, M. G. (1999) Cell Death Differ. 6, Humke, E. W., Shriver, S. K., Starovasnik, M. A., Fairbrother, W. J., and Dixit, V. M. (2000) Cell 103, Chou, J. J., Matsuo, H., Duan, H., and Wagner, G. (1998) Cell 94, Manon, F., Favier, A., Núñez, G., Simorre, J. P., and Cusack, S. (2007) J. Mol. Biol. 365, NOVEMBER 20, 2009 VOLUME 284 NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 32941

Protein Structure Determination Using NMR Restraints BCMB/CHEM 8190

Protein Structure Determination Using NMR Restraints BCMB/CHEM 8190 Protein Structure Determination Using NMR Restraints BCMB/CHEM 8190 Programs for NMR Based Structure Determination CNS - Brünger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-Kunstleve,

More information

PROTEIN'STRUCTURE'DETERMINATION'

PROTEIN'STRUCTURE'DETERMINATION' PROTEIN'STRUCTURE'DETERMINATION' USING'NMR'RESTRAINTS' BCMB/CHEM'8190' Programs for NMR Based Structure Determination CNS - Brünger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-Kunstleve,

More information

Useful background reading

Useful background reading Overview of lecture * General comment on peptide bond * Discussion of backbone dihedral angles * Discussion of Ramachandran plots * Description of helix types. * Description of structures * NMR patterns

More information

Supporting Information

Supporting Information Supporting Information German Edition: DOI: Sampling of Glycan-Bound Conformers by the Anti-HIV Lectin Oscillatoria agardhii agglutinin in the Absence of Sugar** Marta G. Carneiro, Leonardus M. I. Koharudin,

More information

Timescales of Protein Dynamics

Timescales of Protein Dynamics Timescales of Protein Dynamics From Henzler-Wildman and Kern, Nature 2007 Summary of 1D Experiment time domain data Fourier Transform (FT) frequency domain data or Transverse Relaxation Ensemble of Nuclear

More information

Solving the three-dimensional solution structures of larger

Solving the three-dimensional solution structures of larger Accurate and rapid docking of protein protein complexes on the basis of intermolecular nuclear Overhauser enhancement data and dipolar couplings by rigid body minimization G. Marius Clore* Laboratory of

More information

Sequential Assignment Strategies in Proteins

Sequential Assignment Strategies in Proteins Sequential Assignment Strategies in Proteins NMR assignments in order to determine a structure by traditional, NOE-based 1 H- 1 H distance-based methods, the chemical shifts of the individual 1 H nuclei

More information

Timescales of Protein Dynamics

Timescales of Protein Dynamics Timescales of Protein Dynamics From Henzler-Wildman and Kern, Nature 2007 Dynamics from NMR Show spies Amide Nitrogen Spies Report On Conformational Dynamics Amide Hydrogen Transverse Relaxation Ensemble

More information

Supplementary Materials for

Supplementary Materials for advances.sciencemag.org/cgi/content/full/4/1/eaau413/dc1 Supplementary Materials for Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins Per Jemth*, Elin

More information

Theory and Applications of Residual Dipolar Couplings in Biomolecular NMR

Theory and Applications of Residual Dipolar Couplings in Biomolecular NMR Theory and Applications of Residual Dipolar Couplings in Biomolecular NMR Residual Dipolar Couplings (RDC s) Relatively new technique ~ 1996 Nico Tjandra, Ad Bax- NIH, Jim Prestegard, UGA Combination of

More information

Magnetic Resonance Lectures for Chem 341 James Aramini, PhD. CABM 014A

Magnetic Resonance Lectures for Chem 341 James Aramini, PhD. CABM 014A Magnetic Resonance Lectures for Chem 341 James Aramini, PhD. CABM 014A jma@cabm.rutgers.edu " J.A. 12/11/13 Dec. 4 Dec. 9 Dec. 11" " Outline" " 1. Introduction / Spectroscopy Overview 2. NMR Spectroscopy

More information

BMB/Bi/Ch 173 Winter 2018

BMB/Bi/Ch 173 Winter 2018 BMB/Bi/Ch 173 Winter 2018 Homework Set 8.1 (100 Points) Assigned 2-27-18, due 3-6-18 by 10:30 a.m. TA: Rachael Kuintzle. Office hours: SFL 220, Friday 3/2 4:00-5:00pm and SFL 229, Monday 3/5 4:00-5:30pm.

More information

Supporting Information

Supporting Information Supporting Information Ellena et al. 10.1073/pnas.0908317106 SI Experimental Procedures Protein Expression and Sample Preparation. Syb(1 96) and Syb(1 116) from Rattus norvegicus were expressed in BL21(DE3)

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION 5 N 4 8 20 22 24 2 28 4 8 20 22 24 2 28 a b 0 9 8 7 H c (kda) 95 0 57 4 28 2 5.5 Precipitate before NMR expt. Supernatant before NMR expt. Precipitate after hrs NMR expt. Supernatant after hrs NMR expt.

More information

Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2009

Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2009 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2009 Helical Hairpin Structure of a potent Antimicrobial Peptide MSI-594 in Lipopolysaccharide Micelles by NMR Anirban

More information

Supplementary Information

Supplementary Information Electronic Supplementary Material (ESI) for Chemical Communications. This journal is The Royal Society of Chemistry 2017 Supplementary Information Probing the excited-state chemical shifts and exchange

More information

HSQC spectra for three proteins

HSQC spectra for three proteins HSQC spectra for three proteins SH3 domain from Abp1p Kinase domain from EphB2 apo Calmodulin What do the spectra tell you about the three proteins? HSQC spectra for three proteins Small protein Big protein

More information

NMR in Medicine and Biology

NMR in Medicine and Biology NMR in Medicine and Biology http://en.wikipedia.org/wiki/nmr_spectroscopy MRI- Magnetic Resonance Imaging (water) In-vivo spectroscopy (metabolites) Solid-state t NMR (large structures) t Solution NMR

More information

Residual Dipolar Couplings BCMB/CHEM 8190

Residual Dipolar Couplings BCMB/CHEM 8190 Residual Dipolar Couplings BCMB/CHEM 8190 Recent Reviews Prestegard, A-Hashimi & Tolman, Quart. Reviews Biophys. 33, 371-424 (2000). Bax, Kontaxis & Tjandra, Methods in Enzymology, 339, 127-174 (2001)

More information

I690/B680 Structural Bioinformatics Spring Protein Structure Determination by NMR Spectroscopy

I690/B680 Structural Bioinformatics Spring Protein Structure Determination by NMR Spectroscopy I690/B680 Structural Bioinformatics Spring 2006 Protein Structure Determination by NMR Spectroscopy Suggested Reading (1) Van Holde, Johnson, Ho. Principles of Physical Biochemistry, 2 nd Ed., Prentice

More information

Table S1. Primers used for the constructions of recombinant GAL1 and λ5 mutants. GAL1-E74A ccgagcagcgggcggctgtctttcc ggaaagacagccgcccgctgctcgg

Table S1. Primers used for the constructions of recombinant GAL1 and λ5 mutants. GAL1-E74A ccgagcagcgggcggctgtctttcc ggaaagacagccgcccgctgctcgg SUPPLEMENTAL DATA Table S1. Primers used for the constructions of recombinant GAL1 and λ5 mutants Sense primer (5 to 3 ) Anti-sense primer (5 to 3 ) GAL1 mutants GAL1-E74A ccgagcagcgggcggctgtctttcc ggaaagacagccgcccgctgctcgg

More information

Supplementary Figure 1.

Supplementary Figure 1. a b c d e f g 1 Supplementary Figure 1. Identification of unfolded regions in the Chz1-H2A.Z-H2B complex and structure and dynamics of Chz.core-sH2B_H2A.Z. (a) 1 H- 15 N HSQC spectrum of Chz1. All backbone

More information

Introduction solution NMR

Introduction solution NMR 2 NMR journey Introduction solution NMR Alexandre Bonvin Bijvoet Center for Biomolecular Research with thanks to Dr. Klaartje Houben EMBO Global Exchange course, IHEP, Beijing April 28 - May 5, 20 3 Topics

More information

T 1, T 2, NOE (reminder)

T 1, T 2, NOE (reminder) T 1, T 2, NOE (reminder) T 1 is the time constant for longitudinal relaxation - the process of re-establishing the Boltzmann distribution of the energy level populations of the system following perturbation

More information

Nature Structural & Molecular Biology: doi: /nsmb.3194

Nature Structural & Molecular Biology: doi: /nsmb.3194 Supplementary Figure 1 Mass spectrometry and solution NMR data for -syn samples used in this study. (a) Matrix-assisted laser-desorption and ionization time-of-flight (MALDI-TOF) mass spectrum of uniformly-

More information

Interpreting and evaluating biological NMR in the literature. Worksheet 1

Interpreting and evaluating biological NMR in the literature. Worksheet 1 Interpreting and evaluating biological NMR in the literature Worksheet 1 1D NMR spectra Application of RF pulses of specified lengths and frequencies can make certain nuclei detectable We can selectively

More information

Structural characterization of NiV N 0 P in solution and in crystal.

Structural characterization of NiV N 0 P in solution and in crystal. Supplementary Figure 1 Structural characterization of NiV N 0 P in solution and in crystal. (a) SAXS analysis of the N 32-383 0 -P 50 complex. The Guinier plot for complex concentrations of 0.55, 1.1,

More information

Biophysical Journal, Volume 96. Supporting Material

Biophysical Journal, Volume 96. Supporting Material Biophysical Journal, Volume 96 Supporting Material NMR dynamics of PSE-4 β-lactamase: an interplay of ps-ns order and μs-ms motions in the active site Sébastien Morin and Stéphane M. Gagné NMR dynamics

More information

Course Notes: Topics in Computational. Structural Biology.

Course Notes: Topics in Computational. Structural Biology. Course Notes: Topics in Computational Structural Biology. Bruce R. Donald June, 2010 Copyright c 2012 Contents 11 Computational Protein Design 1 11.1 Introduction.........................................

More information

Supplemental Information

Supplemental Information Supplemental Information Combinatorial Readout of Unmodified H3R2 and Acetylated H3K14 by the Tandem PHD Finger of MOZ Reveals a Regulatory Mechanism for HOXA9 Transcription Yu Qiu 1, Lei Liu 1, Chen Zhao

More information

Biochemistry 530 NMR Theory and Practice

Biochemistry 530 NMR Theory and Practice Biochemistry 530 NMR Theory and Practice Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington 1D spectra contain structural information.. but is hard to extract:

More information

PROTEIN NMR SPECTROSCOPY

PROTEIN NMR SPECTROSCOPY List of Figures List of Tables xvii xxvi 1. NMR SPECTROSCOPY 1 1.1 Introduction to NMR Spectroscopy 2 1.2 One Dimensional NMR Spectroscopy 3 1.2.1 Classical Description of NMR Spectroscopy 3 1.2.2 Nuclear

More information

Protein Structure. W. M. Grogan, Ph.D. OBJECTIVES

Protein Structure. W. M. Grogan, Ph.D. OBJECTIVES Protein Structure W. M. Grogan, Ph.D. OBJECTIVES 1. Describe the structure and characteristic properties of typical proteins. 2. List and describe the four levels of structure found in proteins. 3. Relate

More information

Evaluation of the Utility of NMR Structures Determined from Minimal NOE-Based Restraints for Structure-Based Drug Design, Using MMP-1 as an Example

Evaluation of the Utility of NMR Structures Determined from Minimal NOE-Based Restraints for Structure-Based Drug Design, Using MMP-1 as an Example Biochemistry 2000, 39, 13365-13375 13365 Evaluation of the Utility of NMR Structures Determined from Minimal NOE-Based Restraints for Structure-Based Drug Design, Using MMP-1 as an Example Xuemei Huang,

More information

File: {ELS_REV}Cavanagh X/Revises/Prelims.3d Creator: / Date/Time: /9:29pm Page: 1/26 PREFACE

File: {ELS_REV}Cavanagh X/Revises/Prelims.3d Creator: / Date/Time: /9:29pm Page: 1/26 PREFACE PREFACE The second edition of Protein NMR Spectroscopy: Principles and Practice reflects the continued rapid pace of development of biomolecular NMR spectroscopy since the original publication in 1996.

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION DOI: 10.1038/NCHEM.1299 Protein fold determined by paramagnetic magic-angle spinning solid-state NMR spectroscopy Ishita Sengupta 1, Philippe S. Nadaud 1, Jonathan J. Helmus 1, Charles D. Schwieters 2

More information

Protein NMR. Bin Huang

Protein NMR. Bin Huang Protein NMR Bin Huang Introduction NMR and X-ray crystallography are the only two techniques for obtain three-dimentional structure information of protein in atomic level. NMR is the only technique for

More information

NMR, X-ray Diffraction, Protein Structure, and RasMol

NMR, X-ray Diffraction, Protein Structure, and RasMol NMR, X-ray Diffraction, Protein Structure, and RasMol Introduction So far we have been mostly concerned with the proteins themselves. The techniques (NMR or X-ray diffraction) used to determine a structure

More information

Supporting Information

Supporting Information Supporting Information Micelle-Triggered b-hairpin to a-helix Transition in a 14-Residue Peptide from a Choline-Binding Repeat of the Pneumococcal Autolysin LytA HØctor Zamora-Carreras, [a] Beatriz Maestro,

More information

Protein dynamics from NMR Relaxation data

Protein dynamics from NMR Relaxation data Protein dynamics from NMR Relaxation data Clubb 3/15/17 (S f2 ) ( e ) Nitrogen-15 relaxation ZZ-exchange R 1 = 1/T 1 Longitudinal relaxation (decay back to z-axis) R 2 = 1/T 2 Spin-spin relaxation (dephasing

More information

Introduction to Comparative Protein Modeling. Chapter 4 Part I

Introduction to Comparative Protein Modeling. Chapter 4 Part I Introduction to Comparative Protein Modeling Chapter 4 Part I 1 Information on Proteins Each modeling study depends on the quality of the known experimental data. Basis of the model Search in the literature

More information

Acta Crystallographica Section D

Acta Crystallographica Section D Supporting information Acta Crystallographica Section D Volume 70 (2014) Supporting information for article: Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling

More information

Introduction to" Protein Structure

Introduction to Protein Structure Introduction to" Protein Structure Function, evolution & experimental methods Thomas Blicher, Center for Biological Sequence Analysis Learning Objectives Outline the basic levels of protein structure.

More information

Sequential resonance assignments in (small) proteins: homonuclear method 2º structure determination

Sequential resonance assignments in (small) proteins: homonuclear method 2º structure determination Lecture 9 M230 Feigon Sequential resonance assignments in (small) proteins: homonuclear method 2º structure determination Reading resources v Roberts NMR of Macromolecules, Chap 4 by Christina Redfield

More information

Computational Protein Design

Computational Protein Design 11 Computational Protein Design This chapter introduces the automated protein design and experimental validation of a novel designed sequence, as described in Dahiyat and Mayo [1]. 11.1 Introduction Given

More information

Interleukin-1 Receptor Antagonist Protein: Solution Secondary Structure from NOE's and 1H«and 13C«Chemical Shifts

Interleukin-1 Receptor Antagonist Protein: Solution Secondary Structure from NOE's and 1H«and 13C«Chemical Shifts 202 Bulletin of Magnetic Resonance Interleukin-1 Receptor Antagonist Protein: Solution Secondary Structure from NOE's and 1H«and 13C«Chemical Shifts Brian J. Stockman, Terrence A. Scahill, Annica Euvrard,

More information

Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets

Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets Supporting information Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets Wan-Na Chen, Christoph Nitsche, Kala Bharath Pilla, Bim Graham, Thomas

More information

NMR BMB 173 Lecture 16, February

NMR BMB 173 Lecture 16, February NMR The Structural Biology Continuum Today s lecture: NMR Lots of slides adapted from Levitt, Spin Dynamics; Creighton, Proteins; And Andy Rawlinson There are three types of particles in the universe Quarks

More information

Accurate Characterisation of Weak Protein- Protein Interactions by Titration of NMR Residual Dipolar Couplings

Accurate Characterisation of Weak Protein- Protein Interactions by Titration of NMR Residual Dipolar Couplings Accurate Characterisation of Weak Protein- Protein Interactions by Titration of NMR Residual Dipolar Couplings Jose Luis Ortega-Roldan, Malene Ringkjøbing Jensen*, Bernhard Brutscher, Ana I. Azuaga, Martin

More information

Principles of NMR Protein Spectroscopy. 2) Assignment of chemical shifts in a protein ( 1 H, 13 C, 15 N) 3) Three dimensional structure determination

Principles of NMR Protein Spectroscopy. 2) Assignment of chemical shifts in a protein ( 1 H, 13 C, 15 N) 3) Three dimensional structure determination 1) Protein preparation (>50 aa) 2) Assignment of chemical shifts in a protein ( 1 H, 13 C, 15 N) 3) Three dimensional structure determination Protein Expression overexpression in E. coli - BL21(DE3) 1

More information

Orientational degeneracy in the presence of one alignment tensor.

Orientational degeneracy in the presence of one alignment tensor. Orientational degeneracy in the presence of one alignment tensor. Rotation about the x, y and z axes can be performed in the aligned mode of the program to examine the four degenerate orientations of two

More information

Solution Structure and Backbone Dynamics of the TGFβ Type II Receptor Extracellular Domain,

Solution Structure and Backbone Dynamics of the TGFβ Type II Receptor Extracellular Domain, 026 Biochemistry 2003, 42, 026-039 Solution Structure and Backbone Dynamics of the TGFβ Type II Receptor Extracellular Domain, Shashank Deep, Kerfoot P. Walker, III, Zhanyong Shu, and Andrew P. Hinck*,

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Figure S1. Secondary structure of CAP (in the camp 2 -bound state) 10. α-helices are shown as cylinders and β- strands as arrows. Labeling of secondary structure is indicated. CDB, DBD and the hinge are

More information

Experimental Techniques in Protein Structure Determination

Experimental Techniques in Protein Structure Determination Experimental Techniques in Protein Structure Determination Homayoun Valafar Department of Computer Science and Engineering, USC Two Main Experimental Methods X-Ray crystallography Nuclear Magnetic Resonance

More information

Protein Dynamics. The space-filling structures of myoglobin and hemoglobin show that there are no pathways for O 2 to reach the heme iron.

Protein Dynamics. The space-filling structures of myoglobin and hemoglobin show that there are no pathways for O 2 to reach the heme iron. Protein Dynamics The space-filling structures of myoglobin and hemoglobin show that there are no pathways for O 2 to reach the heme iron. Below is myoglobin hydrated with 350 water molecules. Only a small

More information

Spin Relaxation and NOEs BCMB/CHEM 8190

Spin Relaxation and NOEs BCMB/CHEM 8190 Spin Relaxation and NOEs BCMB/CHEM 8190 T 1, T 2 (reminder), NOE T 1 is the time constant for longitudinal relaxation - the process of re-establishing the Boltzmann distribution of the energy level populations

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:10.1038/nature17991 Supplementary Discussion Structural comparison with E. coli EmrE The DMT superfamily includes a wide variety of transporters with 4-10 TM segments 1. Since the subfamilies of the

More information

Molecular Modeling lecture 2

Molecular Modeling lecture 2 Molecular Modeling 2018 -- lecture 2 Topics 1. Secondary structure 3. Sequence similarity and homology 2. Secondary structure prediction 4. Where do protein structures come from? X-ray crystallography

More information

Table S1. Overview of used PDZK1 constructs and their binding affinities to peptides. Related to figure 1.

Table S1. Overview of used PDZK1 constructs and their binding affinities to peptides. Related to figure 1. Table S1. Overview of used PDZK1 constructs and their binding affinities to peptides. Related to figure 1. PDZK1 constru cts Amino acids MW [kda] KD [μm] PEPT2-CT- FITC KD [μm] NHE3-CT- FITC KD [μm] PDZK1-CT-

More information

Deuteration: Structural Studies of Larger Proteins

Deuteration: Structural Studies of Larger Proteins Deuteration: Structural Studies of Larger Proteins Problems with larger proteins Impact of deuteration on relaxation rates Approaches to structure determination Practical aspects of producing deuterated

More information

Protein Structure Prediction II Lecturer: Serafim Batzoglou Scribe: Samy Hamdouche

Protein Structure Prediction II Lecturer: Serafim Batzoglou Scribe: Samy Hamdouche Protein Structure Prediction II Lecturer: Serafim Batzoglou Scribe: Samy Hamdouche The molecular structure of a protein can be broken down hierarchically. The primary structure of a protein is simply its

More information

Using NMR to study Macromolecular Interactions. John Gross, BP204A UCSF. Nov 27, 2017

Using NMR to study Macromolecular Interactions. John Gross, BP204A UCSF. Nov 27, 2017 Using NMR to study Macromolecular Interactions John Gross, BP204A UCSF Nov 27, 2017 Outline Review of basic NMR experiment Multidimensional NMR Monitoring ligand binding Structure Determination Review:

More information

Atomic structure and handedness of the building block of a biological assembly

Atomic structure and handedness of the building block of a biological assembly Supporting Information: Atomic structure and handedness of the building block of a biological assembly Antoine Loquet, Birgit Habenstein, Veniamin Chevelkov, Suresh Kumar Vasa, Karin Giller, Stefan Becker,

More information

Macromolecular X-ray Crystallography

Macromolecular X-ray Crystallography Protein Structural Models for CHEM 641 Fall 07 Brian Bahnson Department of Chemistry & Biochemistry University of Delaware Macromolecular X-ray Crystallography Purified Protein X-ray Diffraction Data collection

More information

antibodies, it is first necessary to understand the solution structure antigenic human epithelial mucin core peptide. The peptide EXPERIMENTAL

antibodies, it is first necessary to understand the solution structure antigenic human epithelial mucin core peptide. The peptide EXPERIMENTAL Biochem. J. (1990) 267, 733-737 (Printed in Great Britain) Elements of secondary structure in a human epithelial mucin core peptide fragment Saul J. B. TENDLER Department of Pharmaceutical Sciences, University

More information

Introduction to biomolecular NMR spectroscopy

Introduction to biomolecular NMR spectroscopy Oct 2002 Introduction to biomolecular NMR spectroscopy Michael Sattler, Structural & Computational Biology EMBL Heidelberg Contents Introduction...2 History... 3 Methodological developments for structure

More information

Biochemistry 530 NMR Theory and Practice. Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington

Biochemistry 530 NMR Theory and Practice. Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington Biochemistry 530 NMR Theory and Practice Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington 1D spectra contain structural information.. but is hard to extract:

More information

Slow symmetric exchange

Slow symmetric exchange Slow symmetric exchange ϕ A k k B t A B There are three things you should notice compared with the Figure on the previous slide: 1) The lines are broader, 2) the intensities are reduced and 3) the peaks

More information

Physiochemical Properties of Residues

Physiochemical Properties of Residues Physiochemical Properties of Residues Various Sources C N Cα R Slide 1 Conformational Propensities Conformational Propensity is the frequency in which a residue adopts a given conformation (in a polypeptide)

More information

Guided Prediction with Sparse NMR Data

Guided Prediction with Sparse NMR Data Guided Prediction with Sparse NMR Data Gaetano T. Montelione, Natalia Dennisova, G.V.T. Swapna, and Janet Y. Huang, Rutgers University Antonio Rosato CERM, University of Florance Homay Valafar Univ of

More information

Molecular structure and dynamics of proteins in solution: Insights derived from high-resolution NMR approaches*

Molecular structure and dynamics of proteins in solution: Insights derived from high-resolution NMR approaches* Pure Appl. Chem., Vol. 75, No. 10, pp. 1371 1381, 2003. 2003 IUPAC Molecular structure and dynamics of proteins in solution: Insights derived from high-resolution NMR approaches* Dennis A. Torchia and

More information

NMR in Structural Biology

NMR in Structural Biology NMR in Structural Biology Exercise session 2 1. a. List 3 NMR observables that report on structure. b. Also indicate whether the information they give is short/medium or long-range, or perhaps all three?

More information

Structural and mechanistic insight into the substrate. binding from the conformational dynamics in apo. and substrate-bound DapE enzyme

Structural and mechanistic insight into the substrate. binding from the conformational dynamics in apo. and substrate-bound DapE enzyme Electronic Supplementary Material (ESI) for Physical Chemistry Chemical Physics. This journal is the Owner Societies 215 Structural and mechanistic insight into the substrate binding from the conformational

More information

BMB/Bi/Ch 173 Winter 2018

BMB/Bi/Ch 173 Winter 2018 BMB/Bi/Ch 173 Winter 2018 Homework Set 8.1 (100 Points) Assigned 2-27-18, due 3-6-18 by 10:30 a.m. TA: Rachael Kuintzle. Office hours: SFL 220, Friday 3/2 4-5pm and SFL 229, Monday 3/5 4-5:30pm. 1. NMR

More information

Structurele Biologie NMR

Structurele Biologie NMR MR journey Structurele Biologie MR 5 /3C 3 /65 MR & Structural biology course setup lectures - Sprangers R & Kay LE ature (27) basics of MR (Klaartje ouben: k.houben@uu.nl; 4/2) from peaks to data (ans

More information

Fast reconstruction of four-dimensional NMR spectra from plane projections

Fast reconstruction of four-dimensional NMR spectra from plane projections Journal of Biomolecular NMR 28: 391 395, 2004. KLUWER/ESCOM 2004 Kluwer Academic Publishers. Printed in the Netherlands. 391 Fast reconstruction of four-dimensional NMR spectra from plane projections Eriks

More information

Errors in the Measurement of Cross-Correlated Relaxation Rates and How to Avoid Them

Errors in the Measurement of Cross-Correlated Relaxation Rates and How to Avoid Them Journal of Magnetic Resonance 44, 8 87 () doi:.6/jmre..56, available online at http://www.idealibrary.com on Errors in the Measurement of Cross-Correlated Relaxation Rates and How to Avoid Them T. Carlomagno

More information

Protein Structure Basics

Protein Structure Basics Protein Structure Basics Presented by Alison Fraser, Christine Lee, Pradhuman Jhala, Corban Rivera Importance of Proteins Muscle structure depends on protein-protein interactions Transport across membranes

More information

Supersecondary Structures (structural motifs)

Supersecondary Structures (structural motifs) Supersecondary Structures (structural motifs) Various Sources Slide 1 Supersecondary Structures (Motifs) Supersecondary Structures (Motifs): : Combinations of secondary structures in specific geometric

More information

CAP 5510 Lecture 3 Protein Structures

CAP 5510 Lecture 3 Protein Structures CAP 5510 Lecture 3 Protein Structures Su-Shing Chen Bioinformatics CISE 8/19/2005 Su-Shing Chen, CISE 1 Protein Conformation 8/19/2005 Su-Shing Chen, CISE 2 Protein Conformational Structures Hydrophobicity

More information

Quantification of Dynamics in the Solid-State

Quantification of Dynamics in the Solid-State Bernd Reif Quantification of Dynamics in the Solid-State Technische Universität München Helmholtz-Zentrum München Biomolecular Solid-State NMR Winter School Stowe, VT January 0-5, 206 Motivation. Solid

More information

Supplementary Material

Supplementary Material Supplementary Material 4D APSY-HBCB(CG)CDHD experiment for automated assignment of aromatic amino acid side chains in proteins Barbara Krähenbühl 1 Sebastian Hiller 2 Gerhard Wider 1 1 Institute of Molecular

More information

Basics of protein structure

Basics of protein structure Today: 1. Projects a. Requirements: i. Critical review of one paper ii. At least one computational result b. Noon, Dec. 3 rd written report and oral presentation are due; submit via email to bphys101@fas.harvard.edu

More information

Using cryoprobes to decrease acquisition times of triple-resonance experiments used for protein resonance assignments

Using cryoprobes to decrease acquisition times of triple-resonance experiments used for protein resonance assignments Spectroscopy 17 (2003) 161 167 161 IOS Press Using cryoprobes to decrease acquisition times of triple-resonance experiments used for protein resonance assignments Michael J. Goger a,, James M. McDonnell

More information

Major Types of Association of Proteins with Cell Membranes. From Alberts et al

Major Types of Association of Proteins with Cell Membranes. From Alberts et al Major Types of Association of Proteins with Cell Membranes From Alberts et al Proteins Are Polymers of Amino Acids Peptide Bond Formation Amino Acid central carbon atom to which are attached amino group

More information

Protein Structure Determination using NMR Spectroscopy. Cesar Trinidad

Protein Structure Determination using NMR Spectroscopy. Cesar Trinidad Protein Structure Determination using NMR Spectroscopy Cesar Trinidad Introduction Protein NMR Involves the analysis and calculation of data collected from multiple NMR techniques Utilizes Nuclear Magnetic

More information

1) NMR is a method of chemical analysis. (Who uses NMR in this way?) 2) NMR is used as a method for medical imaging. (called MRI )

1) NMR is a method of chemical analysis. (Who uses NMR in this way?) 2) NMR is used as a method for medical imaging. (called MRI ) Uses of NMR: 1) NMR is a method of chemical analysis. (Who uses NMR in this way?) 2) NMR is used as a method for medical imaging. (called MRI ) 3) NMR is used as a method for determining of protein, DNA,

More information

Supporting Protocol This protocol describes the construction and the force-field parameters of the non-standard residue for the Ag + -site using CNS

Supporting Protocol This protocol describes the construction and the force-field parameters of the non-standard residue for the Ag + -site using CNS Supporting Protocol This protocol describes the construction and the force-field parameters of the non-standard residue for the Ag + -site using CNS CNS input file generatemetal.inp: remarks file generate/generatemetal.inp

More information

Details of Protein Structure

Details of Protein Structure Details of Protein Structure Function, evolution & experimental methods Thomas Blicher, Center for Biological Sequence Analysis Anne Mølgaard, Kemisk Institut, Københavns Universitet Learning Objectives

More information

Use of deuterium labeling in NMR: overcoming a sizeable problem Michael Sattler and Stephen W Fesik*

Use of deuterium labeling in NMR: overcoming a sizeable problem Michael Sattler and Stephen W Fesik* Ways & Means 1245 Use of deuterium labeling in NMR: overcoming a sizeable problem Michael Sattler and Stephen W Fesik* Address: Abbott Laboratories, 47G AP10,100, Abbott Park Road, Abbott Park, IL 60064-3500,

More information

Dihedral Angles. Homayoun Valafar. Department of Computer Science and Engineering, USC 02/03/10 CSCE 769

Dihedral Angles. Homayoun Valafar. Department of Computer Science and Engineering, USC 02/03/10 CSCE 769 Dihedral Angles Homayoun Valafar Department of Computer Science and Engineering, USC The precise definition of a dihedral or torsion angle can be found in spatial geometry Angle between to planes Dihedral

More information

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease University of Wollongong Research Online Faculty of Science - Papers (Archive) Faculty of Science, Medicine and Health 2009 NMR study of complexes between low molecular mass inhibitors and the West Nile

More information

NMR Characterization of Partially Folded and Unfolded Conformational Ensembles of Proteins

NMR Characterization of Partially Folded and Unfolded Conformational Ensembles of Proteins Elisar Barbar Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701 NMR Characterization of Partially Folded and Unfolded Conformational Ensembles of Proteins Abstract: Studies of

More information

Secondary and sidechain structures

Secondary and sidechain structures Lecture 2 Secondary and sidechain structures James Chou BCMP201 Spring 2008 Images from Petsko & Ringe, Protein Structure and Function. Branden & Tooze, Introduction to Protein Structure. Richardson, J.

More information

Biophysical Chemistry: NMR Spectroscopy

Biophysical Chemistry: NMR Spectroscopy Relaxation & Multidimensional Spectrocopy Vrije Universiteit Brussel 9th December 2011 Outline 1 Relaxation 2 Principles 3 Outline 1 Relaxation 2 Principles 3 Establishment of Thermal Equilibrium As previously

More information

Residual Dipolar Couplings Measured in Multiple Alignment Media.

Residual Dipolar Couplings Measured in Multiple Alignment Media. Residual Dipolar Couplings Measured in Multiple Alignment Media. We have already seen that the orientational degeneracy inherent to a single measured coupling can be raised by measuring different directions

More information

Crystal Structure of Fibroblast Growth Factor 9 (FGF9) Reveals Regions. Implicated in Dimerization and Autoinhibition

Crystal Structure of Fibroblast Growth Factor 9 (FGF9) Reveals Regions. Implicated in Dimerization and Autoinhibition JBC Papers in Press. Published on November 1, 2000 as Manuscript M006502200 Crystal Structure of Fibroblast Growth Factor 9 (FGF9) Reveals Regions Implicated in Dimerization and Autoinhibition 1 Copyright

More information

Solution Structure of ZipA, a Crucial Component of Escherichia coli Cell Division

Solution Structure of ZipA, a Crucial Component of Escherichia coli Cell Division 9146 Biochemistry 2000, 39, 9146-9156 Solution Structure of ZipA, a Crucial Component of Escherichia coli Cell Division Franklin J. Moy, Elizabeth Glasfeld, Lidia Mosyak, and Robert Powers*, Department

More information

SUPPLEMENTARY FIGURES

SUPPLEMENTARY FIGURES SUPPLEMENTARY FIGURES Supplementary Figure 1 Protein sequence alignment of Vibrionaceae with either a 40-residue insertion or a 44-residue insertion. Identical residues are indicated by red background.

More information

Protein Structure Determination from Pseudocontact Shifts Using ROSETTA

Protein Structure Determination from Pseudocontact Shifts Using ROSETTA Supporting Information Protein Structure Determination from Pseudocontact Shifts Using ROSETTA Christophe Schmitz, Robert Vernon, Gottfried Otting, David Baker and Thomas Huber Table S0. Biological Magnetic

More information