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Efficient NMR Sequential Assignment Strategies for Protein Backbone Analysis

Learn sequential resonance assignment strategies for accurate protein analysis, including backbone and side chain assignments, NOE contacts, and tertiary structure determination using NMR techniques. Understand how to place residues in sequence, identify resonances, infer protein fold, and enrich assignments with NOE data for precise structural calculations. Explore practical issues, medium-range NOEs, heteronuclear assignments, and more in this comprehensive guide.

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Efficient NMR Sequential Assignment Strategies for Protein Backbone Analysis

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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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