1. Introduction to biological NMR Dominique Marion Institut de Biologie Structurale Grenoble France

2. Presentation outline  Structural investigation by NMR  NMR spectral parameters  The NMR spectrometer  Two dimensional NMR  Protein HSQC  NMR resonance assignment  NMR structure calculation  Protein-ligand interaction  Molecular motion and relaxation

3. Structural investigations by NMR (1) Sample preparation (a) Optimization of the bacterial expression (b) Optimization of the protein expression (c) Labelling [ 15 N] or [ 15 N- 13 C] or [ 15 N- 13 C- 2 H] (2) NMR experiment recording (a) Preliminary 2D experiments to optimize experimental conditions (b) 2D homonuclear experiments (< 80 aa) or 3D triple resonance experiments (>80 aa) (3) Sequential resonance assignment (a) Backbone resonances (b) Side-chain resonances

4. Structural investigations by NMR (4) Collection of structural restraints (a) Internuclear distances (nOe) (b) Dihedral angles (J-coupling) (c) Internuclear vector orientations (RDC) (5) Structure calculation and refinement (a) Simulated annealing (b) Structure refinement (MD simulation) (c) Structure validation (NMR statistics) (6) Complementary studies (a) Protein dynamics (Relaxation and echange) (b) Interaction with partners (ligands…)

5. NMR spectral parameters nOe RDC Line-width ShieldingChemical shift Scalar interactionJ-coupling Nuclear Overhauser effectDipolar interaction Residual dipolar coupling Relaxation

6. NMR spectral parameters Line-width J (Hz) J-coupling J+D (Hz) RDC nOe Nuclear Overhauser effect 0  (ppm) Chemical shift

7. Chemical shift: ring current Upfield shifted resonance Downfield shifted resonance

8. J-coupling (scalar coupling) J (Hz) AX Nucleus Electronic cloud Nuclear spin Electronic spin H —— C 1 J CH > 0

9. J-couplings in 15 N 13 C labelled proteins 1 J C’N =15Hz 1 J NC  =11Hz 1 J C  C  =35Hz 1 J C  C’ =55Hz 1 J NH =92Hz 1 J C  H =140Hz 2 J NC  =7Hz 2 J NC’ < 1Hz

10. Nuclear Overhauser effect Relaxation in NMR:  processes that allow the magnetization to return to equilibrium Origin:  modulation of a spin interaction by the molecular motion Relaxation mechanisms in NMR:  Dipole-dipole interaction  Chemical shift anisotropy nOe (Nuclear Overhauser effect)  Transfer of nuclear magnetization from I to S via dipolar cross-relaxation I S r IS B0B0 

11. Nuclear Overhauser effect Energy diagram for a two-spin system. The levels are populated according to a Boltzman distribution law. A radiofrequency field saturates the A transitions: The corresponding populations are equalized. In small molecules with a fast tumbling rate, the transition at high frequency W 2 is efficient. A population increase is observed for spin A. In large molecules with a slow tumbling Rate, the transition at low frequency W 0 is efficient. A population decrease is observed for spin A.

12. I S r IS B0B0  The sign and strength of the dipolar coupling interaction between I and S depends on the relative orientation of the nuclei with respect to B 0. Residual dipolar coupling Isotropic solutionWeakly aligned medium All orientation of the IS vector are equally likely. The dipolar coupling averages to zero. No structural information The proteins become weakly aligned. The dipolar coupling does not average exactly to zero. RDC structural information

13. Residual dipolar coupling Alignment tensor Describes the preferential orientation of the protein Measured RDCs Depends upon the orientation of the internuclear vector with respect to the alignment tensor.

14. B0B0 J-coupling vs RDC J-couplings provide information on the relative orientation of the two internuclear vectors RDCs provide information on the absolute orientation of each internuclear vector with respect to a common molecular reference frame

15. Experimental measurement of J-coupling and nOe Signal presaturation before spectrum recording Continuous irradiation during spectrum recording

16. NMR spectrometer Superconducting magnet NMR console Rf generation and amplification Workstation Spectrometer control NMR probe

17. Superconducting magnet Dewar Insulation Liquid nitrogen Liquid helium Main magnet coil Magnet legs NMR detection probe Sample

18. Two-dimensional NMR [1] Jean Jeener, AMPERE Summer School in Basko Polje, Yugoslavia, September 1971 PreparationMixingEvolutionDetection t1t1 t2t2 The preparation and the mixing period do not change during the experiment.

19. Two-dimensional NMR [2] PreparationMixing Evolution Detection t2t2  t1t1 t2t2  t 1 t2t2  t 1 t2t2 The receiver is open during the detection but not during the evolution

20. Two-dimensional NMR [3] t1t1 t2t2 Along t 2, all the data points are recorded in real time. Along t 1, each data point requires a new experiment.

21. t1t1 t2t2 Two-dimensional NMR [4] Strong signal at the beginning Weak signal at the end (thermal noise)

22. Two-dimensional NMR [5] Fourier transform along the rowsFourier transform along the columns t1t1 t2t2 t2t2 t1t1

23. Two-dimensional NMR [5] t2t2 t1t1 F2F2 F1F1 F2F2 t1t1

24. Two-dimensional NMR [6] Chemical reaction A + X B + Y Step 2: chemical reaction AB More frequently: equilibrium reaction

25. Two-dimensional NMR [7] Correlation spectroscopy PreparationMixingEvolutionDetection t1t1 t2t2 Step 0: preparation of the reactants 0 Step 1: identification of the reactants 1 Step 2: chemical reaction 2 Step 3: identification of the products 3

26. Two-dimensional NMR [8] Correlation spectroscopy AB PreparationMixingEvolutionDetection t1t1 t2t2 F1F1 F2F2 A B AB AB A A B A B B Diagonal peaks Cross-peaks

27. Two-dimensional NMR [9] 1D NMR signal (in the absence of relaxation) The NMR signal is always described as a complex number 2D NMR signal cos sin x y z

28. Two-dimensional NMR [10] Amplitude modulation 2D NMR signal Cos (  1 t 1 ) Cos (  2 t 2 )Cos (  1 t 1 ) Sin (  2 t 2 ) Sin (  1 t 1 ) Sin (  2 t 2 )Sin (  1 t 1 ) Cos (  2 t 2 ) RRRI IRII Hypercomplex data

29. Two-dimensional NMR [11] 2D NMR signal Cos (  1 t 1 ) Cos (  2 t 2 )Cos (  1 t 1 ) Sin (  2 t 2 )Sin (  1 t 1 ) Sin (  2 t 2 )Sin (  1 t 1 ) Cos (  2 t 2 ) RRRI IRII Quadrature detection (States Method) PreparationMixingEvolutionDetection t1t1 t2t2 Prep +x Prep +y

30. 1 H- 15 N correlation spectrum of a protein 1D cross-section along the 1 H dimension 1D cross-section along the 15 N dimension

31. 1 H- 15 N correlation spectrum of a protein Folded protein 175 residue imipenem-acylated L,D- transpeptidase from B. subtilis Lecoq et al Structure 20, 850-861 (2012). Disordered protein 179 residue fragment of hepatitis C virus non-structural protein 5A Feuerstein et al Biomol. NMR Assign. 5, 241-243 (2011). Glycine residues

32. NMR resonance assignment Goal: Connecting a nucleus in the proteina resonance in the spectrum

33. The useful information is not the absolute position of a piece …. But the connectivity with its neighbors. The jigsaw puzzle analogy for NMR resonance assignment Two pieces have been already successfully matched Their shape fits roughly the profile of the already matched pair But only one piece could be anchored effortlessly This strategy is repeated for all future candidates. When the puzzle is nearly complete, the location of the remaining pieces can be easily deduced… Two pieces are possible candidates as neighbors on the right hand side

34. NMR resonance assignment

36. Once the resonance assignement has been obtained, the location of the secondary structure elements (  -helices and  -sheets) can be determined … without computing the complete NMR structure. Protein secondary structure prediction TALOS + : Empirical prediction of protein [  ] backbone torsion angles using HN, HA, CA, CB, CO, N chemical shift assignments   Secondary structure elements in the computed structure  -helices

37. NMR structure calculations [1] Collecting conformational restraints Distance restraints nOe between nearby hydrogens Possible pitfalls and difficulties: – multi-spin effect or spin diffusion – conformational averaging (missing nOe) – required distance calibration Long-range and small nOe carry more structural information Separation into 3 different classes: – strong nOe (< 2.8 Å) – medium nOe ( < 3.4Å) – small nOe

38. NMR structure calculations [2] Collecting conformational restraints Dihedral angles Vicinal 3 J coupling constant Karplus relationship Chemical shifts allow the identification of secondary structure elements Chemical shift index (CSI method) /Talos Finding a suitable alignment medium Protein solubility / possible alteration of the conformation Residual dipolar coupling

39. NMR structure calculations [3] Traditional approach for structure calculation (a)Collecting assigned structural information (b) Start from a random conformation (c) Restrained molecular dynamic with a simplified force field. (d) Refinement of the structure with a complete force field and water molecules. Automated methods for structure calculation Automated NOESY assignment during structure calculation

40. NMR structure calculations [4] Automated methods for structure calculation

41. NMR structure calculations [5] Disordered N- and C-termini Disordered loop Bacillus subtilis l,D-Transpeptidase 169 amino-acids Ribbon representation  -sheets  -helices

42. NMR and Refinement Statistics for NMR Structures Total NOE3,191 Intraresidue1,479 Interresidue1,712 Sequential (|i – j| = 1) 681 Medium-range (|i – j | < 4) 325 Long-range (|i – j| > 5) 706 Total dihedral angle restraints286  143  143 Total RDC169 NH85 C  H  84 Qualitative RDC agreement (%)17 Lecoq L et al. 2012. Dynamics Induced by  -Lactam Antibiotics in the Active Site of Bacillus subtilis l,D-Transpeptidase. Structure/Folding and Design 20: 850–61. Bacillus subtilis l,D-Transpeptidase 169 amino-acids Bacillus subtilis l,D-Transpeptidase 169 amino-acids

43. Violations (mean and SD) Distance constraints (Å)0.062 ± 0.005 Dihedral angle constraints (º) 1.87 ± 0.03 Max. dihedral angle violation17 Max. distance constraint violation1.61 Deviations from idealized geometry Bond lengths (Å)0.0068 Bond angles (º)0.97 Impropers (º)2.34 Average pairwise rmsd (Å) Heavy0.70 ± 0.10 Backbone 0.39 ± 0.09 Is the calculated structure in agreement with the experimental data? Is the covalent geometry of the polypeptidic chain not distorted? What is the scattering within the set of structures that have been calculated? NMR and Refinement Statistics for NMR Structures Lecoq L et al. 2012. Dynamics Induced by  -Lactam Antibiotics in the Active Site of Bacillus subtilis l,D-Transpeptidase. Structure/Folding and Design 20: 850–61.

44. NMR vs X-rays

45. Protein-ligand interaction Addition of the ligand to the protein sample Observation of the protein spectrum 1D NMR or fast 2D NMR P P PLPL PL P P PLPL P + L PL P + L PL Slow exchange Tight binding Fast exchange Weak binding

46. Protein-ligand interaction The ligand is added to the protein: Some chemical shift variations are observed on the protein.They are located primarily at the binding interface A paramagnetic tag is attached to the ligand Line-broadenings are observed on the protein at the binding interface. Nuclear Overhauser effect can be observed between nuclei in the protein and in the ligand. Discrimination of intra- and intermolecular nOe is possible by means of isotopic labelling. Residual dipolar couplings can be measured for The two partners and the complex. If differences are observed, they can be explained by changes in the preferential orientation of the 2 molecules

47. Protein dynamics by NMR Protein function  important role of the flexibility Protein dynamics = time dependent-fluctuations over a wide range of time scale. Ligand binding Catalytic enzymes Folding pathways Aggregation Thermostability Molten globule Misfolding Conformational entropy Excited states

48. NMR observables and protein motions 10 -12 10 -9 10 -6 10 -3 110 3 T1, T2, nOe RDC CMPG EXSY RT NMR Side chain rotation Protein global tumbling Protein folding Enzymatic reactions Ligand binding Nuclear spin relaxationRelaxation dispersion Real-time NMR Time (sec)

49. Inverted population What is NMR relaxation? Boltzmann equilibrium Magnetization recovery 1 – 2 exp(-t/T 1 ) Longitudinal relaxation time

50. Molecular motion and relaxation Molecular motions in the liquid-state: Global molecular tumbling Internal fluctuations (side-chains, domains) I S r IS B0B0  Molecular motions modulate the spin interactions Here the dipolar interaction between spin I and S  The fluctuations of the spin interaction create a local fluctuating magnetic field. This fluctuating magnetic fields push the magnetization toward its equilibrium. M z =M z 0 and M x =M y =0

51. Molecular motion and relaxation C ––– H cc ii S2S2 NMR relaxation provides information: On the speed of the molecular rotation On the speed of internal motions On the amplitude of internal motions

52. LinkerTailsModules Molecular motion and relaxation Order parameter (S 2 ) Protein sequence 1.0 0.8 0.0 Protein made of two domains connected by a small linker

53. Presentation outline  Structural investigation by NMR  NMR spectral parameters  The NMR spectrometer  Two dimensional NMR  Protein HSQC  NMR resonance assignment  NMR structure calculation  Protein-ligand interaction  Molecular motion and relaxation