Site-directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance (EPR) Spectroscopy. An Introduction

Transcription

1 Site-directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance (EPR) Spectroscopy An Introduction Johann P. Klare Department of Physics, University of Osnabrück, Osnabrück, Germany 1

2 Outline EPR Spectroscopy A short introduction What is a spin label? Spin labeling of proteins: the classical approach EPR-Tools: What can we learn from spin labeled biomolecules Spin-Spin Distance Determination Spin Labeling Techniques A general protocol for spin labeling using cysteine-specific reagents 2

3 Electron Paramagnetic Resonance (EPR) or Electron Spin Resonance (ESR) What is it? EPR is the resonance spectroscopy of molecular systems with an unpaired electron. 3

4 Does my system have unpaired electrons?... Organic Radicals in proteins amino acid radicals (tyrosine...) protein bound cofactor radicals (semiquinones, flavines) myoglobin cytochrome bc1 complex Metal centres in proteins /protein complexes Cu, Mn, Ni, Co, Mo, Fe hemes, FeS clusters Short-lived radicals / Reactive Oxygen Species (ROS) Spin Traps / Spin Probes O cytochrome c oxidase 4

5 Why Spin labelling? (Most) proteins and nucleic acids don t have unpaired electrons! no EPR?!...but... we can introduce the wanted unpaired electron into almost any system under investigation and we can do this also (almost) wherever we want Site-directed spin labeling (SDSL) 5

6 Does my system have unpaired electrons?... Organic Radicals in proteins amino acid radicals (tyrosine...) protein bound cofactor radicals (semiquinones, flavines) myoglobin cytochrome bc1 complex Metal centres in proteins /protein complexes Cu, Mn, Ni, Co, Mo, Fe hemes, FeS clusters Spin labels attached to proteins or nucleic acids Short-lived radicals / Reactive Oxygen Species (ROS) Spin Traps / Spin Probes O cytochrome c oxidase 6

7 The Electron Spin Elementary particles such as an electron (and also atomic nuclei!) are characterized by an intrinsic angular momentum: The Spin S! they behave like spinning tops Elementary particles are quantum particles the rules of quantum mechanics apply! The Spin can be in two states, which differ in the orientation of the angular momentum. m S = ½ & m S = - ½ The Spin makes the electron behave like a tiny magnet! 7

8 The Electron Spin in a Magnetic Field: The Concept of Magnetic Resonance without a magnetic field, the two spin states of the electron are degenerate they have the same energy if we place the electron in a magnetic field, the two spin states will have different energies (the energy levels are separated by DE). Zeeman Effect or Electron-Zeeman Effect undisturbed electron in an external magnetic field B 8

9 The Resonance Condition First: What does resonance mean? (Microwave) radiation is used for the transition of molecules from one state to the other: lower state higher state (absorption) higher state lower state (stimulated emission) Normally, we have more molecules (n 0 ) in the (energetically) lower state (ground state) than in the higher state (n 1 ) (excited state) according to the Boltzmann distribution n 1 = n 0 exp(-de/kt) Resonance results in net absorption of radiation. The Resonance condition for a two-level system in EPR is hn = g b B k = Boltzmann constant (1, J/K) Energy of the radiation Energy difference between the two molecular states produced by B. Frequency n (or often angular frequency w = 2p n), for which the resonance condition is fulfilled: Larmor Frequency 9

10 EPR signal We can explain our first EPR spectrum! DPPH hn = g b B n 9.5 GHz (X band) Field / Gauss In most EPR machines the 1 st derivative of the absorption spectrum is recorded! ( Field Modulation) DPPH (Diphenylpikrylhydrazyl) A common standard for field calibration in EPR g = /

11 ...most EPR spectra have more then one line... another submolecular magnet comes into play: the nucleus with its nuclear spin I Multiplicity: M I = 2I + 1 Biological transition metal ions Metal Isotope Spin Multiplicity Mn 55 5/2 6 Fe 54,56,57, 58 1/2 (2%) 2 Co 59 7/2 8 Cu 63, 65 3/2 4 Mo 92,94,95, 96,97,98, 100 5/2 6 Ligand atoms Ligand Isotope Spin Multiplicity H 1, 2 1/2, 1 (0.02%) 2, 3 C 12, 13 1/2 (1.1%) 2 N 14, 15 1, 1/2 3, 2 O 16,17,18 5/2 6 P 31 1/2 2 Cl 35,27 3/2 4 Multiplicity = number of EPR lines 11

12 Types of Interactions EPR spectra can reflect many different magnetic interactions, giving rise to (sometimes complicated) multi-line spectra....but (good news)... We can describe and analyse most of the spectra just by considering pairwise interactions between magnets of three types: 1. electron spins S 2. nuclear spins I 3. laboratory magnets B...and (even better news)... This results in five basic types of interactions, from which two are usually so weak, that we can ignore them! 12

13 Types of Interactions electron spins nuclear spins laboratory magnets S I B Interaction Phenomenon Example S x B Zeeman Interaction Basic EPR Pattern S x I S x S Hyperfine Interactions Zero-Field Interactions Dipolar Interactions Metal hyperfine (e.g. Cu (I=3/2)) Ligand hyperfine ( 1 H (I=½), 14 N (I=1)) I x I Quadrupole Interaction Mn (I = 5/2) I x B Nuclear Zeeman Interaction Double Resonance Spectra (ENDOR) and NMR!! 13

14 Interaction of the Electron Spin with Nuclear Spins: Hyperfine Interaction S x I The electron experiences not just one, but M I different types of nuclei (with different local magnetic fields!). Each type causes its own shift in the EPR resonance line Splitting into M I EPR lines! Hyperfine interaction for a Nitroxide radical (S = ½, I = 1) A Hyperfine constant Multiplicity: M I = 2I

15 Types of Interactions electron spins nuclear spins laboratory magnets S I B Interaction Phenomenon Example S x B Zeeman Interaction Basic EPR Pattern S x I S x S Hyperfine Interactions Zero-Field Interactions Dipolar Interactions Metal hyperfine (e.g. Cu (I=3/2)) Ligand hyperfine ( 1 H (I=½), 14 N (I=1)) I x I Quadrupole Interaction Mn (I = 5/2) I x B Nuclear Zeeman Interaction Double Resonance Spectra (ENDOR) and NMR!! 15

16 Dipolar Interactions S x S A not so simple equation (the Hamiltonian describing the dipole-dipole interaction)......with a simple message: The dipolar interaction between two electrons is proportional to r -3, r being the distance between the two electrons. ^ ^ S 1 and S 2 : spin operators for electrons 1 and 2. Quantification of the dipolar interaction allows us to determine the distance between two paramagnetic centers!...what is dipolar interaction?...how to quantify?... 16

17 Dipolar Interactions S x S...what is dipolar interaction? resonance frequency of spin 1 without dipolar interaction angular frequency w (at a fixed B field position) hn = g b B w = 2p n ħw = g b B 17

18 Dipolar Interactions...what is dipolar interaction? resonance frequency of spin 1 with dipolar interaction with parallel aligned spin 2 angular frequency w (at a fixed B field position) hn = g b B w = 2p n S x S ħw = g b B dipolar interaction with parallel spin 2 B field at spin 1 is decreased lower resonance frequency 18

19 Dipolar Interactions...what is dipolar interaction? resonance frequency of spin 1 with dipolar interaction with antiparallel aligned spin 2 angular frequency w (at a fixed B field position) hn = g b B w = 2p n S x S ħw = g b B dipolar interaction with antiparallel spin 2 B field at spin 1 is increased higher resonance frequency 19

20 Dipolar Interactions...what is dipolar interaction? dipolar frequency w dd S x S angular frequency w (at a fixed B field position) hn = g b B w = 2p n ħw = g b B w dd (r) r -3 20

21 Outline EPR Spectroscopy A short introduction What is a spin label? Spin labeling of proteins: the classical approach EPR-Tools: What can we learn from spin labeled biomolecules Spin-Spin Distance Determination Spin Labeling Techniques Proteins Nucleic Acids A general protocol for spin labeling using cysteine-specific reagents 21

22 What is a spin label?...a stable chemical compound which possesses an unpaired electron (i.e. it is a stable radical) and a specific reactive group to be bound to (bio)molecules. The vast majority of spin labels are nitroxides, where the unpaired electron is located at an NO group, which is usually part of a heterocyclic ring. Functional groups contained within the spin label allow them to be attached to the molecule under investigation and determine their specificity. e.g.: Protein thiol groups specifically react with the functional groups methanethiosulfonate, maleimide, and iodoacetamide, creating a covalent bond with cysteine. A little bit of history: Spin labels were first synthesized in the laboratory of H. M. McConnell in The first spin labeling studies have been performed using thiol-specific functional groups to label natively occuring cysteines in proteins (e.g. in hemoglobin). Site-directed spin labeling (SDSL) was pioneered in the laboratory of Dr. W.L. Hubbell in the late 80 s/early 90 s. 22

23 Site-directed spin labeling of proteins: the classical approach 23

24 SDSL-EPR-Tools: Overview 1. Spin Label Mobility 2. Accessibility towards paramagnetic quencher molecules (NiEDDA, CrOx, O 2 ) Discrimination between lipid bilayer, aqueous phase and protein interior 3. Polarity of the SL Microenvironment 4. Transient Structural Changes 5. Spin-Spin Distance determination (~ Å) Information about Structure and Dynamics 24

25 Spin Label Mobility The (RT) EPR spectral shape is sensitive to the reorientational motion of the nitroxide side chain (partial motional averaging of the anisotropic components of the g- and hyperfine tensors). For spin-labeled sites exposed to the bulk water, the nitroxide mobility is slightyl restricted. rotational correlation times in the ns range. small line widths of the center lines, DH 0 small apparent hyperfine splittings. Restricted mobility of the spin label side chain (interaction with neighboring side chains or backbone atoms) line widths and the apparent hyperfine splittings are increased 25

26 Spin Label Mobility The correlation between the inverse linewidth DH 0-1 and the inverse of the spectral second moment H 2-1 allows a general classification of the region where the spin label is located. The spectral second moment H 2 quantifies the breadth of the EPR spectrum. Klare, J.P., Steinhoff, H.-J., Photosynth. Res. 102, (2009) 26

27 Spin Label Mobility - Identification/analysis of multiple components Example: Analysis of a temperature and ionic strength dependent equilibrium between a compact and a dynamic form of a protein domain. The cw EPR spectra show two components (1 and 2) with different rotational correlation times t c. Arrhenius plot (logarithm of the rotational correlation times of the two components vs. 1/T) van t Hoff plot (natural logarithm of the ratio of the two spectral components vs. 1/T) Doebber et al, J.Biol.Chem..283, (2008) 27

28 Amplitude central line Amplitude central line Amplitude central line Accessibility for Paramagnetic Quencher Molecules O 2 CrOx N2 lipid CrOx O2 N2 water CrOx O2 N2 protein P 1/ P mw 1/ 2 P 1/2 [Relaxing agent]- P 1/2 [N 2 ] [Relaxing agent] [Relaxing agent] Klare, J.P., Steinhoff, H.-J., Photosynth. Res. 102, (2009) 28

29 Polarity of the SL Microenvironment Polarity and proticity of the spin label microenvironment are reflected in the hyperfine component A zz the g tensor component g xx A zz can be readily obtained from X band spectra. A polar environment shifts A zz to higher values g xx to lower values polarity To obtain g xx we need to go to W band (at least). 29

30 Assignment of secondary structures Mobility, Accessibility, Polarity Accessibility Analysis Mobility Analysis PutP Polarity Analysis A Na + /proline symporter 30

31 Monitoring Transient Structural Changes Conformational changes in an archaebacterial photoreceptor/-transducer protein complex upon light excitation. F G TM2 Klare, J.P., Steinhoff, H.-J., and Engelhard, M. (2004) FEBS Lett. 564, 219 Position 159: change of the EPR signal due to transient mobilisation upon outward motion of helix F Position 78 : change of the EPR signal due to increased dipolar interaction between the two spin labels 31

32 Spin-Spin Distance Determination Low Temperature CW EPR Double Electron Electron Resonance (DEER) (1-2 nm) (2-8 nm) Simulation of EPR distance distributions...or how to translate the EPR data into a model of your biomolecule º molecular dynamics (MD) simulations º the rotamer library approach (RLA) 32

33 Spin-Spin Distance Determination Dipolar Interactions dipolar frequency w dd w dd (r) r -3 w broadening of cw EPR spectra 33

34 Low Temperature CW EPR: Steinhoff et al. (1997) Biophys. J. 73,

35 Low Temperature CW EPR: Steinhoff et al. (1997) Biophys. J. 73,

36 Low Temperature CW EPR: Steinhoff et al. (1997) Biophys. J. 73,

37 Pulse Electron Double Resonance (PELDOR) or Double Electron-Electron Resonance (DEER) dipolar frequency w dd w dd (r) r -3 37

38 PELDOR/DEER G domain dimerization of the trna-modifying enzyme MnmE monitored with PELDOR. (Meyer et al., PLoS Biol. 7, e (2009)) 38

39 Spin Labeling Techniques Proteins Spin labeling of cysteines Spin labeling by peptide synthesis Spin labeling using click chemistry Spin labeling using the nonsense suppressor methodology Nucleic Acids Nucleic Acids Usage of spin labeled nucleotide analogs in oligonucleotide synthesis Postsynthetic modification of nucleotide analogs with reactive groups Postsynthetic modification at the sugar/phosphate backbone 39

40 Spin Labeling of Cysteines 1. Methanethiosulfonate spin labels Formation of a disulfide bond: MTSSL sensitivity towards reducing conditions! MTSSL: (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate MTSSL structure MTSSL comprises a flexible linker minimizing disturbances of the native fold of the protein! Size: comparable to that of a tryptophane side chain. Besides MTSSL, a variety of other different nitroxide radical compounds are commercially available, which - have a longer or shorter linker - are sterically more demanding - are ph sensitive -... MTS-4-oxyl spin label 40

41 Spin Labeling of Cysteines 2. Maleimide spin labels Formation of a C-S bond: no sensitivity towards reducing conditions!...but... sterically more demanding (disturbance of protein structure!) can react also with primary amines (N-term., Lysine, Arginine) 3. (Iodo)acetamide spin labels Formation of a C-S bond: no sensitivity towards reducing conditions! long, flexible linker...but... can react also with secondary amines (esp. Histidine) 41

42 Spin Labeling by (solid phase) peptide synthesis (SPPS) The most popular spin label building block is the paramagnetic α-amino acid TOAC (4-amino-1-oxyl-2,2,6,6,-tetramethyl-piperidine-4-carboxylic acid). TOAC exhibits only one degree of freedom: the ring conformation. direct information about the orientation of secondary structure elements! Using the method of (solid phase) peptide synthesis, polypeptides with arbitrary amino acids (incl. spin labeled amino acids!) can be synthesized. Spin label building blocks for the standard Boc- or Fmoc-based peptide synthesis have been synthesized or are commercially available. Drawback of the method: Peptide synthesis is still limited to < 200 amino acids! 42

43 Spin Labeling by peptide synthesis - Expressed Protein Ligation (EPL) Semisynthesis of proteins from recombinant and synthetic fragments two polypeptide fragments: peptide 1 (recombinant) peptide 2 (synthetic, carrying the spin label) formation of a native peptide bond between the two fragments by chemical ligation. 43

44 Spin Labeling using click chemistry Highly selective formation of a carbon-heteroatom bond under mild conditions ( stability of the nitroxide radical) and with high yield. 1,3-dipolar cycloaddition of organic azides with alkynes in the presence of Cu(I) (copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition,cuaac) 44

45 Spin Labeling of nucleic acids labeling of the nucleobase labeling of the backbone sugar Spin Labelling 45

46 Spin Labeling of nucleic acids 1. Usage of spin labeled nucleotide analogs in oligonucleotide synthesis Chemically synthesized nucleotide analogs are used in standard oligonuleotide synthesis. Example: Spin-labeled thymidine analog, where a methyl group is replaced by an acetylene-tethered nitroxide 2. Postsynthetic modification of nucleotide analogs with reactive groups Usage of nucleotide analogs chemically modified with reactive groups suitable for post-synthetic modification with a spin labeling reagent. Example: 4-thiouridine (for RNA) derivatives, which can be modified with thiol-specific reagent like MTSSL. Spin Labelling 46

47 Postsynthetic modification at the sugar/phosphate backbone Alternatively, the spin label can be attached to the sugar moiety of specific nucleotides Example: Postsynthetic derivatization of a 2 -amino group introduced into oligonucleotides with a isocyanate derivative of a TEMPO-like moiety. Ward et al., ChemBioChem 8, (2007) 47

48 A general protocol for spin labeling using cysteine-specific reagents 1. Spin labeling in solution Incubate the purified protein for 1-2 h with a reducing agent (DTT, DTE) to reduce oxidized thiol groups. Remove the reducing agent completely (!) by an appropriate method (washing in centrifugal concentrators, gel filtration, affinity chromatography (for proteins carrying an affinity tag), desalting columns, dialysis). Use a protective atmosphere (N 2, Ar) to prevent re-oxidation. Adjust the protein concentration be < 500 µm. Incubate the protein with a 2-5fold excess (subject to optimization) of the spin label reagent. (Spin label compounds like MTSSL are usually quite hydrophobic. Stock solutions of 100 mm spin label in e.g. DMSO have been proven to be convenient for most cases.) The duration and the temperature at which incubation is performed depends on the actual system. Incubations over night at 4 C or 2-12h at RT usually lead to good labeling efficiencies for well-accessible labeling sites. (Also this point is subject to optimization) Remove unbound/unreacted spin label. The same procedure as for the removal of the reducing agent could/should be applied. Check the labeling efficiency! Check the functionality of the labeled protein protein/biomolecule!!! 48

49 A general protocol for spin labeling using cysteine-specific reagents 2. Spin labeling on affinity columns Bind the protein to the affinity column. (Spin labelling could be the last step of your protein purification, just before you would elute it from the column) Incubate the purified protein for 1-2 h with a reducing agent (DTT, DTE) to reduce oxidized thiol groups. Wash out the reducing agent completely (!). Incubate the protein with a 2-5fold excess (subject to optimization) of the spin label reagent. (Spin label compounds like MTSSL are usually quite hydrophobic. Stock solutions of 100 mm spin label in e.g. DMSO have been proven to be convenient for most cases.) The duration and the temperature at which incubation is performed depends on the actual system. Incubations over night at 4 C or 2-12h at RT usually lead to good labelling efficiencies for well-accessible labelling sites. (Also this point is subject to optimization) Wash out unbound/unreacted spin label. Elute the protein from the column. Check the labeling efficiency! Check the functionality of the labeled protein protein/biomolecule!!! 49