NMR Scalar and Dipolar Coupling

1. NMR Scalar and Dipolar Coupling Through Space Through Bonds • Coupling of nuclei gives information on structure

2. OH CH2 CH3 The key attribute: use the scalar and dipolar couplings to match the set of signals with the molecular structure Resonance Assignment CH3-CH2-OH Which signal from which H atoms?

3. Proteins Have Many Signals 1H NMR Spectrum of Ubiquitin ~500 resonances • A large number of signals are overlapped

4. Protein NMR:Overlapped Sub-Spectra Each residue in the sequence gives rise to an independent NMR sub-spectrum, which is much simpler than the complete protein spectrum Methods have been developed to extract each sub-spectrum from the whole

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

6. Homonuclear 1H Assignment Strategy • Scalar coupling to identify resonances, dipolar couplings to place in sequence • Based on backbone NH • Concept: build out from the backbone to identify the side chain resonances • 2nd dimension resolves overlaps, 3D rare 1H 1H 1H

7. H H H H H H N—C N—C N—C COSY: One J coupling A B C

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

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

10. 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

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