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Resonance Assignment NMR analysis of proteins Sequential resonance assignment strategies Practical Issues
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Sequential Assignment 1.Assign the protein backbone N, HN, CA, HA, CB, HB, C 2. Assign sidechains 3. Assign NOE contacts
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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
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Put Residues In Sequence ABCDZ Intraresidue Sequential Medium-range (helices) Tertiary Structure Use only these to make sequential assignments
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Sequential NOEs -strand H (i-1) -> HN(i) -helix :H (i-1) -> HN(i)
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NOE based Assignment 15 N edited 1 H- 1 H TOCSY/NOESY
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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
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Heteronuclear Assignments: Backbone Experiments Names of scalar experiments based on atoms detected Acquired in ‘pairs’
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Heteronuclear Assignments: Side Chain Experiments
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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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Identify more NOEs In the course of refinement
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