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

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Presentation on theme: "Resonance Assignment NMR analysis of proteins Sequential resonance assignment strategies Practical Issues."— Presentation transcript:

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)

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

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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’

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16 Heteronuclear Assignments: Side Chain Experiments

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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

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21 Identify more NOEs In the course of refinement


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