1. 1 Biomolecular NMR Spectroscopy Methods and applications to proteins Robert Kaptein Novosibirsk, November 2012

2. 2 Contents  Introduction to Biomolecular NMR  Multidimensional NMR for proteins  Resonance assignments  Observables & structure restraints  3D Structure determination by NMR  Protein Dynamics  Example: Lac repressor, structure, dynamics, DNA interactions

3. 3 Introduction to Biomolecular NMR

4. 4 Elements of Protein NMR

5. Pros & cons of NMR in structural biology Pros... –no need for crystal: no crystal packing artifacts, solution more native- like – study of dynamics: picosecond to seconds time scales, conformational averaging, chemical reactions, folding... –easy study of protein-protein, protein-DNA, protein- ligand interactions Cons... –NMR structure determination is a bit slow.... –Need isotope labeling ( 13 C, 15 N) –solution NMR works best for MW < 50 kDa 5 5

6. NMR & Structural biology Dynamic activation of an allosteric regulatory protein Tzeng S-R & Kalodimos CG Nature (2009) DYNAMICS F-helices Apo-CAP CAP-cAMP

7. 7 NMR & Structural biology TRANSIENT COMPLEXES Visualization of the Encounter Ensemble of the Transient Electron Transfer Complex of Cytochrome c and Cytochrome c Peroxidase Bashir Q. et al JACS (2010)

8. 8 NMR & Structural biology EXCITED STATES Structure and Dynamics of Pin1 During Catalysis by NMR Labeikovsky W. et al JMB (2007)

9. 9 NMR & Structural biology MEMBRANE PROTEINS Mechanisms of Proton Conduction and Gating in Influenza M2 Proton Channels from Solid-State NMR Hu F. et al Science (2010)

10. 10 NMR & Structural biology High-resolution multidimensional NMR spectroscopy of proteins in human cells Inomata K. et al Nature (2009) IN-CELL NMR

11. 11 Nuclear spin (rad. T -1. s -1 )

12. 12 The NMR sample Isotope labeling  15 N for small proteins < 15 kDa  15 N & 13 C for larger proteins, up to 30-40 kDa  15 N, 13 C & 2 H for large proteins > 40 kDa  selective labeling (e.g. only methyl groups) Sample - recombinant expression in E.coli  pure, stable and high concentration ➡ 500 µL of 0.5 mM solution: ~ 5 mg per sample  preferably low salt, low pH

13. 13 Amino acids

14. Amino acids are usually referred to with either a three-letter or a one-letter code: GlycineGlyGHistidineHisH AlanineAlaAProlineProP ValineValVAspartateAspD LeucineLeuLGlutamateGluE IsoleucineIleIAsparagineAsnN SerineSerSGlutamineGlnQ ThreonineThrTLysineLysK PhenylalaninePheFArginineArgR TyrosineTyrYCysteineCysC TryptophaneTrpWMethionineMetM Amino acids

15. 15 Multidimensional NMR for Proteins

16. 16 Sensitivity: Signal to noise (S/N) Resolution: Line separation Protein 1 H NMR spectrum

17. 17 multidimensional NMR experiments  resolve overlapping signals ➡ enables assignment of all signals  encode structural or dynamical information ➡ enables structure determination ➡ enables study of dynamics Why multidimensional NMR

18. 18 2D NMR

19. 19 3D NMR

20. 20 1D single FID of N points FID t1t1 2D N FIDs of N points t2t2 FID t1t1 mixing nD experiment 3D NxN FIDs of N points t2t2 t1t1 mixing t3t3 FID direct dimension indirect dimensions

21. 21 nD experiment direct dimension indirect dimensions 1D 1 FID of N points acquisition t1t1 preparation 2D N FIDs of N points t2t2 t1t1 mixing preparationevolution acquisition 3D NxN FIDs of N points t2t2 t1t1 t3t3 mixing preparationevolution mixing evolution acquisition

22. 22 spin-spin interactions ???? proton Aproton B mixing/magnetization transfer Encoding information

23. 23 NOE (Nuclear Overhauser Effect )  magnetic dipole interaction  through space  distance dependent (1/r 6 )  NOESY -> distance restraints J-coupling interaction  through 1-4 bonds max.  chemical connectivities  TOCSY, COSY -> assignment  conformation dependent Encoding information: mixing

24. 24 t2t2 FID t1t1 NOESY tmtm magnetic dipole interaction crosspeak intensity ~1/r 6 up to 5-6 Å COSY t2t2 FID t1t1 J-coupling interaction transfer over one J-coupling, i.e. max. 3-4 bonds TOCSY t2t2 FID t1t1 J-coupling interaction transfer over several J- couplings, i.e. multiple steps over spin-coupled network mlev Homonuclear 1 H NMR

25. 25 NOE effect Dipolar interaction  cross-relaxation  transient NOE build-up  proportional to 1/r 6 t2t2 FID t1t1 NOESY tmtm |αα> |αβ> |βα> |ββ> WIWI WIWI WSWS WSWS W2W2 W0W0

26. 26 t2t2 FID t1t1 tmtm A A (ω A ) A B B (ω B ) F1 F2 ωAωA ωAωA ωBωB proton A proton B ~Å NOESY (F1,F2) = ω A, ω A (F1,F2) = ω A, ω B Diagonal Cross-peak

27. 27 Uses dipolar interaction (NOE) to transfer magnetization between protons ➡ cross-peak intensity ~ 1/r 6 ➡ distances (r) < 5Å NOESY (or 2D NOE) diagonal HNHN HNHN cross-peak

28. Homonuclear scalar coupling

29. COSY: based on 3 J HH

30. COSY & TOCSY

31. COSY o TOCSY *

32. 32  isotope labeling (expression in E.coli)  measure frequencies of different nuclei; e.g. 1 H, 15 N, 13 C  no diagonal peaks  mixing only via J 1H1H 15 N Heteronuclear NMR

33. 33 HSQC (heteronuclear single quantum coherence) t2t2 FID t1t1 1H1H DEC 15 N 1H1H 15 N ( ω 15 N ) 1 J NH (F 1,F 2 ) = ω 15 N, ω 1 H J-mix block Heteronuclear NMR J-mix block 1 H ( ω 1 H )

34. 34 Heteronuclear magnetization transfer INEPT  Insensitive Nuclei Enhanced Polarization Transfer  Enhancement factor: γ H /γ X (i.e. 10 for X = 15 N)  Magnetization transfer through J-coupling  Chemical shift refocussed 1H1H 15 N Δ = 1/(4J HN ) x Δ Δ xxy x

35. 35 Product operator formalism Pulse (x) I z cosα + I y sinα Chemical shift evolution I y cos(ω I t) + I x sin(ω I t) J-coupling evolution I y cos(πJ IS t) + 2I x S z sin(πJ IS t) cos 2 α + sin 2 α = 1 cos 2 α - sin 2 α = cos2α 2cosαsinα = sin2α γB 1 IzIz α USEFUL RULES Clockwise rotations !!! x y z

36. 36 15 N-HSQC

37. 37 Heteronuclear magnetization transfer Refocused INEPT 1H1H 15 N Δ = 1/(4J HN ) Δ Δ y ΔΔ

38. 38 15 N HSQC  Backbone NH  Side-chain NH and NH 2

39. 39 1 H- 15 N HSQC: ‘protein fingerprint’

40. 40 1 H- 15 N HSQC: ‘protein fingerprint’

41. 41 3D NMR Double resonance  Two 1 H frequency axes and one heteronuclear axis ➡ 3D NOESY- 15 N-HSQC ➡ 3D NOESY- 13 C-HSQC ➡ 3D TOCSY- 15 N-HSQC ➡.... Triple-resonance  Three different frequency axes (i.e. 1 H, 15 N and 13 C) ➡ HNCA ➡ HNCACB ➡ HNCO ➡....

42. 42 3 H N atoms same δH N Same δH N different δ 15 N 1H1H 1HN1HN 3D TOCSY-[ 1 H- 15 N]-HSQC 15 N 1 H → 1 H N → 15 N → 1 H N t1t1 t2t3 TOCSY F1F1 F3F3 F2F2 FT HSQC

43. 43 15 N 1HN1HN 1H1H [ 1 H, 15 N]-HSQC projection strip 3D TOCSY-[ 1 H- 15 N]-HSQC

44. 44 HNCA t3t3 FID t2t2 1H1H DEC 15 N (F 1,F 2,F 3 )= (ω 13 Ca(i), ω 15 N(i), ω 1 H(i) ) & (ω 13 Ca(i-1), ω 15 N(i), ω 1 H(i) ) t1t1 13 C (aliphatic) 1H1H 15 N 1 J NH 1 J NCa(i) 2 J NCa(i-1) Triple resonance NMR J-mix block 15 N ( ω 15 N ) 13 C( ω 13 C ) 1 H ( ω 1 H ) J-mix block 1 J NCa(i) 2 J NCa(i-1) 1 J NH J-mix block

45. 45 HNCA pulse sequence

46. 46 Sequential assignment strips of 3D HNCA spectrum ( 15 N dimension ⊥ to screen) 13 Cα (i-1) 13 Cα (i) 1 H N (i)

47. 47 resolve overlapping signals mixing/magnetization transfer NOESY, TOCSY, COSY HSQC 3D double resonance (3D NOESY-HSQC, 3D TOCSY-HSQC) 3D triple resonance (e.g. HNCA) Key concepts multidimensional NMR