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Resonance Assignment

Resonance Assignment. NMR analysis of proteins Sequential resonance assignment strategies Practical Issues. Sequential Assignment. 1.Assign the protein backbone N, HN, CA, HA, CB, HB, C 2. Assign sidechains 3. Assign NOE contacts. Assignment Strategy.

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Resonance Assignment

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

  4. A B C D • • • • Z Put Residues In Sequence Use only these to make sequential assignments Tertiary Structure Sequential Intraresidue Medium-range (helices)

  5. Sequential NOEs a-helix :HN(i-1) -> HN(i) b-strand Ha(i-1) -> HN(i)

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

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

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

  9. Heteronuclear Assignments:Side Chain Experiments

  10. Determining Protein FoldIn Structure Calculations • 1. Determine secondary structure • CSI directly from assignments • Medium-range NOEs • 2. Add key long-range NOEs to fold

  11. Identify more NOEs In the course of refinement

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