1. NMR: Practicalities and Applications BIOC 530

2. Learning Goals Have a better understanding of NMR data in publications Determine how/ if NMR can be useful in your project

3. 3D structures by solution NMR SH3 domain ensemble: Nature Methods 7, S42 - S55 (2010) Why use NMR to solve structures?

4. 3D structures by solution NMR Steps: 1.Over-expression, isotope enrichment, and purification of the protein of interest; 2.Sample optimization of buffer, salt, and pH to maximize protein solubility and minimize oligomerization; 3.Assignment of side-chain and backbone NMR resonances; 4.Determination of structural constraints: NOE (Nuclear Overhauser Enhancement) cross-relaxation rate constants), J-coupling constants, RDCs (residual dipolar couplings), and isotropic chemical shifts; 5.Structure calculation and refinement; structure validation.

5. 1) Protein sample preparation Overwhelming majority of the proteins studied by NMR are over-expressed in and purified from E. Coli M9 (minimal media) with 13 C-enriched glucose and 15 N- enriched ammonium chloride as sole carbon and nitrogen sources is used for 13 C/ 15 N labeling E. Coli growth in D 2 O is used to introduce deuterium into non-exchangeable protein sites. Partial deuteration is useful for NMR studies of proteins > 25 kDa Insect cell medium and in-vitro translation systems enriched with stable isotopes are available; but still prohibitively expensive

6. 2) Optimization of sample conditions Buffers with non-negligible temperature dependence of pH (e.g. Tris) should be avoided. pH < 7 is preferred, as it minimizes the loss of 1 H sensitivity due to exchange with water protons. The protein must be in a well-defined oligomeric state 0.5 - 1 mM is the optimum protein concentration for structural and dynamical studies The NMR sample should be stable over periods of time required to collect the NMR data –days > binding studies –weeks > assignments or dynamics –months > all atom assignments / full dynamics characterization

7. 3) Assignments 4)Structural restraints in NMR Short Distance Restraints –NOEs (from crosspeak intensities in NOESY spectra) Dihedral angle restraints/ secondary structure –J-coupling constants, Chemical Shifts Long Distance Restraints –Residual dipolar couplings (RDCs), paramagnetic spin labels Hydrogen Bond restraints –Amide (NH) hydrogen/deuterium exchange rates, temperature dependence

8. NOE distance constraints: 1 H NOESY spectra Cross-peaks NOE cross-peak intensities relate to the distance between 1 H in space. Can also be done with filtering to obtain 15 NH or 13 CH NOEs etc.

9. Structural restraints: NOEs Initial rate approximation: NOE cross-peak intensities are proportional to the cross-relaxation rate constants Cross-relaxation rate constants are proportional to (1/r 6 ), where r is the distance between the two 1 H that are close in space tyrosine  distance always the same = 2.5 Å r i = r ref (S ref /S i ) 1/6 where S i and S ref are the volumes of cross- peaks measured in NOE spectra. “ref” stands for the reference proton pair with a well- defined geometry H H

10. Short distances found in protein structures

11. Structural restraints: NOEs Hundreds of NOEs are required to obtain a good NMR structure You don’t want to use the exact distance calculated as a restraint. Instead provide a range. classrestraintdescription*for protein w/ M r <20 kDa strong1.8-2.7 Åstrong intensity in short mixing time NOESY medium1.8-3.3 Åweak intensity in short mixing time NOESY weak1.8-5.0 Åonly visible in longer mixing time NOESY

12. Structural constraints: Dihedral angles from J-coupling constants

13. Horst Joachim Schirra's PPS2 project

14. Measuring 3 J HN-H  : 3D HNHA spectra HN to H  crosspeak HN diagonal peak this is one plane of a 3D spectrum of ubiquitin. The plane corresponds to this 15 N chemical shift ratio of crosspeak and diagonal peak intensities can be related to 3 J HN-H  J small J large Archer et al. J. Magn. Reson. 95, 636 (1991).

15. Structural restraints – another option for secondary structure CSI (chemical shift index) - establishes the secondary structure of proteins based on chemical shift differences with respect to some predefined “random coil” values. It can be applied from the measured HA, CA, CB and CO chemical shifts for each residue in a protein. 0 = random coil chemical shift

16. Structural restraints: bond orientations Residual dipolar couplings (RDCs) 1.Intrinsic anisotropy 2.External liquid crystalline medium (sterics and/or charge) Bicelles Phage Polyacrylamide gels C 12 E 5 PEG + hexanol

17. Structural restraints: RDCs Measured for a pair of covalently-linked NMR-active nuclei in partially aligned molecules Examples: 15 N- 1 H, 13 C  - 15 N, 13 CO- 15 N RDCs RDCs depend on the orientation of the bond vector relative to the molecular alignment frame Aligned sample splitting = J NH +D NH N H r B θ 4  r NH 3 ħ  N  H D NH = (1 – 3 cos 2  )

18. PREs long distance restraints – 15-24Å Chem. Rev. 2009, 109, 4108–4139 Paramagnetic DNA or Membrane

19. NMR data do not uniquely define the 3D structures, because the restraints are included as a range of allowed values A good “ensemble” of structures minimizes violations of the input restraints and the RMSD between the members of the ensemble Step 5: Structure calculation and refinement

20. NMR experimental data NMR experimental data Structure ensemble Structure ensemble Validated structure data Validated structure data Experimental restraints Experimental restraints Structure calculation and selection Restraint violation and error analysis Assignment and conversion Structure quality checks and statistics “Validation of NMR-derived protein structures”, Chris Spronk, Centre for Molecular and Biomolecular Informatics, University of Nijmegen, The Netherlands. Step 5: Structure calculation and refinement

21. Ensembles of NMR structures The precision of an NMR structure is determined by the root-mean-square deviation (RMSD) between the backbone atoms of the conformational ensemble NMR structure of RNA-binding SAM (sterile alpha motif) protein, from Edwards et al, J. Mol. Biol. (2006), 356, 1065-1072. Stereo view of the conformational ensemble Stereo view of representative structures

22. Making structural models with limited NMR data CS Rosetta - Makes empirical correlations gained from mining chemical shift data deposited in the BMRB (Biological Magnetic Resonance Bank) database structure prediction based on chemical shift data, chemical shift perturbations, PREs, RDCs, SAXS using ROSETTA Fragment based modeling based on known structures in PDB

23. Limited data refinement example from a zinc coordinating kinase regulatory domain Conformation a RDC (Hz) Conformation b RDC (Hz) Aligned sample splitting = J NH +D NH N H r B θ 4  r NH 3 ħ  N  H D NH = (1 – 3 cos 2  )

24. Limited data refinement example from a zinc coordinating kinase regulatory domain

25. NMR is good for more than just structure determination!

26. Frequency (Hz) k ex =k 1 + k -1 Timescales of binding in NMR k ex <<  Slow exchange k ex >>  Fast exchange k ex =  k -1 k1k1 AB

27. Titration of a membrane bound second messenger, diacylglycerol, into a signaling protein Wild-type signaling protein Fast exchange Tighter binding mutant slow exchange

28. Titration of a membrane bound second messenger, diacylglycerol, into a signaling protein Wild-type signaling protein Fast exchange Tighter binding mutant slow exchange

29. pH dependent conformational exchange Protonation = fast Conformational exchanage = slow

30. Double-stranded RNA binding innate immune protein

31. cAMPfisetin Carlson et al. (2013) Studying ligand binding in a large unassigned protein cAMP Met 572 Voltage gated K + channel (HCN2) Heart - pace making Brain - chronic pain Two activating ligands

32. 13 C-HSQC resonances

33. 13 C-HSQC methyls

34. 13 C-HSQC of HCN2 M572 Carlson et al. (2013)

35. Assignment by mutagenesis Carlson et al. (2013) M572T

36. Met Scanning Technique- Structure 20: 4, p573–581, 4 April 2012

37. Time scales for protein dynamics Librations Side Chain Rotations CPMG, R 1  Folding s 10 -12 10 -9 10 -6 10 -3 10 0 R 1, R 2, NOE, Cross-correlation 1 H exchange Loop and Domain Motions Magnetization transfer Lineshape analysis Atomic resolution Multiple timescales Enzyme Catalysis

38. NMR relaxation parameters Steady-state hetero-nuclear NOE Transverse (or spin-spin) relaxation rate constant, R 2 –Loss of phase coherence in the transverse plane Longitudinal (or spin-lattice) relaxation rate constant, R 1 –Relaxation to Z plane All 3 depend on the motion of your protein in solution –Size/ shape –Hydrodynamic radius –Unstructured regions/ multiple domain proteins B0B0 z y x ω = γB 0

39. Relaxation Dispersion Experiments R 2 =R 2 0 +R ex cpmg 2 + k ex 2 k ex p A p B Δω AB 2 R ex = k ex = kinetic rate constant = k 1 + k -1 p A & p B = populations of A and B Δω AB 2 = difference in chemical shift Characterization of conformational exchange = R ex k -1 k1k1 AB Invisable Population cpmg

40. Relaxation dispersion experiments The information about microsecond- millisecond protein dynamics is buried in R ex. By fitting relaxation dispersion curves one can extract the ratio of exchanging populations, chemical shift differences (  ) between the exchanging conformers and kinetic rate constants (k ex ). cpmg 2 + k ex 2 k ex p A p B Δω AB 2 R ex =

41. Protein dynamics in enzymatic catalysis Substrate binding and product release are often accompanied by conformationalchanges that could be rate-limiting for the catalytic reaction. The catalytic step itself is dynamic, as it involves fluctuations of atomic coordinates during bond breaking or formation. Examples of some of the protein systems where dynamics proved to be crucial for catalysis: dihydrofolate reductase, cyclophilin A, and adenylate kinase.

42. Dynamic energy landscape of DHFR Boehr et al, Science 313, 1638 (2006) There are 5 known intermediates; the structures of these intermediates (or their models) have been determined by X-ray crystallography. DHF THF

43. Conformational changes during the DHFR catalytic cycle

44. Relaxation dispersion data for each DHFR intermediate Red: active site conformation; blue: cofactor binding; and green: substrate/product binding

45. Correlation between the chemical shift data obtained from relaxation dispersion analysis with that of ground state conformations From fitting relaxation dispersion curves Difference in 15 N shifts of ground state from spectra

46. Dynamic energy landscape of DHFR From fitting relaxation dispersion curves From enzyme kinetics assays

47. Reference: Boehr et al, Science 313, 1638 (2006) Librations Side Chain Rotations CPMG, R 1  Folding s 10 -12 10 -9 10 -6 10 -3 10 0 R 1, R 2, NOE, Cross-correlation 1 H exchange Loop and Domain Motions Magnetization transfer Lineshape analysis

48. Solid-state NMR

49. Solid-state NMR: advantages Isotropic-like NMR spectra with site resolution No solubility problem No “tumbling time” problem

50. Kaliotoxin-K + channel interactions The chemical shifts of kaliotoxin are perturbed as a result of binding to K + channel. K + channel kaliotoxin Lange et al, Nature (2006), 440, 959-962

51. Kaliotoxin-K + channel interactions Solid-state structure of kaliotoxin bound to K + channel Residues whose chemical shifts are perturbed as a result of binding are colored red. Lange et al, Nature (2006), 440, 959-962

52. Kaliotoxin-K + channel interactions: looking at K + channel Perturbed and unperturbed residues of K + channel are shown in red and blue, respectively. K + channel kaliotoxin Lange et al, Nature (2006), 440, 959-962

53. Structural model of kaliotoxin-K+ channel High-affinity binding of kaliotoxin is accompanied by an insertion of K27 side-chain into the selectivity filter of the channel; The binding is associated with conformational changes in both molecules. kaliotoxin K + channel selectivity filter Lange et al, Nature (2006), 440, 959-962

54. A couple good reviews for your reference An introduction to NMR-based approaches for measuring protein dynamics: Biochimica et Biophysica Acta 1814 (2011) 942–968 Mapping Protein-Protein Interactions in Solution by NMR Spectroscopy: Biochemistry 41:1 (2002)