Biomolecular Nuclear Magnetic Resonance Spectroscopy

1. 01/26/04 Biomolecular Nuclear Magnetic Resonance Spectroscopy FROM ASSIGNMENT TO STRUCTURE Sequential resonance assignment strategies NMR data for structure determination Structure calculations Properties of NMR structures

2. T L R S G G S Basic Strategy to Assign Resonances in a Protein • Identify resonances for each amino acid • Put amino acids in order - Sequential assignment (R-G-S,T-L-G-S) - Sequence-specific assignment 1 2 3 4 5 6 7 R - G - S - T - L - G - S

3. Homonuclear 1H Assignment Strategy • Scalar coupling to identify resonances, dipolar couplings to place in sequence • Based on backbone NH (unique region of spectrum, greatest dispersion of resonances, least overlap) • Concept: build out from the backbone to identify the side chain resonances • 2nd dimension resolves overlaps, 3D rare 1H 1H 1H

4. Step 1: Identify Spin System

5. H H H H H H N—C N—C N—C COSY: One coupling A B C

6. H H H H H H H H R-COSY: Add A 2nd Coupling N—C—C A N—C—C B N—C—CH3 C

7. H H H H H H H H H H DR-COSY: Add A 3rd Coupling N—C—C A N—C—C B N—C—CH3 C

8. H H H H H H H H H H H H N—C—C—C—C—C—NH3 H H H H H H TOCSY: All Coupled Spins N—C—C—C—COOH A B N—C—CH3 C

9. Peaks in NOESY spectra Same as scalar coupling peaks Peaks from residue i to i+1 A  B (B  C) Step 2: Fit Residues in Sequence A - B - C

10. A B C D • • • • Z A Minor Problem With NOESYMany Types of NOEs Use only these to make sequential assignments Long Range Sequential Intraresidue Medium-range (helices)

11. Extended Homonuclear 1H Strategy • Same basic idea as 1H strategy: based on backbone NH • Concept: when backbone 1H overlaps  disperse with backbone 15N • Use Het. 3D to increase signal resolution 1H 1H 15N

12. 3 overlapped NH resonances with different side chains 15N Dispersed 1H-1H TOCSY Add a 3rd dimension separating out HN overlaps by their 15N frequency

13. 3 overlapped NH resonances Same NH, different 15N F3 F2 F1 TOCSY HSQC 1H 1H 15N t1 t2 t3 15N Dispersed 1H-1H TOCSY

14. Heteronuclear (1H,13C,15N) Strategy • One bond at a time - all atoms (except O) • Even handles backbone 15N1H overlaps  disperse with backbone C’CaHaCbHb… • Het. 3D/4D increases signal resolution 1H  13C 15N 1H • Works on bigger proteins because one bond scalar couplings are larger

15. Heteronuclear Assignments:Backbone Experiments Names of scalar experiments based on atoms detected Consecutive residues!! NOESY not needed

16. Heteronuclear Assignments:Side Chain Experiments Multiple redundancies increase reliability Tutorial on the website

17. Heteronuclear Strategy: Key Points • Bonus: amino acid identification and sequential assignments all at once • Most efficient, but expts. more complex • Enables study of much larger proteins (TROSY/CRINEPT  1 MDa: e.g. Gro EL) • Requires 15N, 13C, [2H] enrichment • High expression in minimal media (E. coli) • Extra $ ($150/g 13C-glucose, $20/g 15NH4Cl)

18. Structure Determination by NMR

19. NMR Experimental Observables Providing Structural Information • Backbone conformation from chemical shifts (Chemical Shift Index- CSI): , • Distance restraints from NOEs • Hydrogen bond restraints • Backbone and side chain dihedral angle restraints from scalar couplings • Orientation restraints from residual dipolar couplings

20. A B C D • • • • Z 1H-1H Distances From NOEs Long-range (tertiary structure) Sequential Intraresidue Medium-range (helices) Challenge is to assign all peaks in NOESY spectra

21. 1H-1H NOESY 2D 1H 1H 3D 1H 1H 1H 1H 1H 1H 1H 1H 1H 1H 1H 15N 13C 13C 15N 15N 15N 13C 13C 1H 1H 3D 4D Approaches to Identifying NOEs • 15N- or 13C-dispersed 1H-1H NOESY

22. 1H 1H Labeled protein Unlabeled peptide 13C Only NOEs at the interface • Transferred NOE:based on 1) faster build-up of NOEs in large versus small molecules; 2) Fast exchange 3) NOEs of bound state detected at resonance frequencies of free state H kon H koff H H Only NOEs from bound state Special NOESY Experiments • Filtered, edited NOE:based on selection of NOEs from two molecules with unique labeling patterns.

23. C=O H-N Backbone Hydrogen Bonds • NH chemical shift at low field (high ppm) • Slow rate of NH exchange with solvent • Characteristic pattern of NOEs • (Scalar couplings across the H-bond) • When H-bonding atoms are known  can impose a series of distance/angle constraints to enforce standard H-bond geometries

24. • • • • 6 Hz Dihedral Angles FromScalar Couplings • Must accommodate multiple solutions multiple J values • But database shows few occupy higher energy conformations

25. F3 F2 F1 Orientational Constraints From Residual Dipolar Couplings (RDCs) Ho Reports angle of inter-nuclear vector relative to magnetic field Ho 1H 1H 13C 15N 1H 1H 15N 1H • Requires medium to partially align molecules • Must accommodate multiple solutions multiple orientations

26. NMR Structure Calculations • Objective is to determine all conformations consistent with the experimental data • Programs that only do conformational search lead to bad chemistry  use molecular force fields improve molecular properties • Some programs try to do both at once • Need a reasonable starting structure • NMR data is not perfect: noise, incomplete data  multiple solutions (conformational ensemble)

27. Variable Resolution of Structures • Secondary structures well defined, loops variable • Interiors well defined, surfaces more variable • Trends the same for backbone and side chains • More dynamics at loops/surface • Constraints in all directions in the interior

28. Restraints and Uncertainty • Large # of restraints = low values of RMSD • Large # of restraints for key hydrophobic side chains

29. Assessing the Qualityof NMR Structures • Number of experimental constraints • RMSD of structural ensemble (subjective!) • Violation of constraints- number, magnitude • Molecular energies • Comparison to known structures: PROCHECK • Back-calculation of experimental parameters Read the book chapter!