1. Resonance Assignment NMR analysis of proteins Sequential resonance assignment strategies Practical Issues

2. Sequential Assignment 1.Assign the protein backbone N, HN, CA, HA, CB, HB, C 2. Assign sidechains 3. Assign NOE contacts

3. Assignment Strategy 1.Identify resonances for each amino acid 2.Put amino acids in order - Sequential assignment (R-G-S,T-L-G-S) 3.Place fragments in aa sequence - Sequence-specific assignment

4. Put Residues In Sequence ABCDZ Intraresidue Sequential Medium-range (helices) Tertiary Structure Use only these to make sequential assignments

5. Sequential NOEs  -strand H  (i-1) -> HN(i)  -helix :H  (i-1) -> HN(i)

7. NOE based Assignment 15 N edited 1 H- 1 H TOCSY/NOESY

9. Large Scalar Couplings  Less Sensitive to MW of the Protein Superior to 1 H homonuclear strategy: H-H couplings <20 Hz Mixing is faster so less time for signal to relax

10. Heteronuclear Assignments: Backbone Experiments Names of scalar experiments based on atoms detected Acquired in ‘pairs’

16. Heteronuclear Assignments: Side Chain Experiments

19. Determining Protein Fold In Structure Calculations 1. Determine secondary structure CSI directly from assignments Medium-range NOEs 2. Add key long-range NOEs to fold

21. Identify more NOEs In the course of refinement