Resonance Assignment

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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

• 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

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Put Residues In Sequence
Use only these to make sequential assignments
Tertiary Structure
Sequential
Intraresidue
Medium-range (helices)
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Sequential NOEs
a-helix HN(i-1) -gt HN(i)
b-strand Ha(i-1) -gt HN(i)
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NOE based Assignment
15N edited 1H-1H TOCSY/NOESY
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Large Scalar Couplings ? Less Sensitive to MW of
the Protein
Superior to 1H homonuclear strategy H-H
couplings lt20 Hz Mixing is faster so less time
for signal to relax
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Heteronuclear AssignmentsBackbone Experiments
Names of scalar experiments based on atoms
detected Acquired in pairs
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Heteronuclear AssignmentsSide Chain Experiments
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Determining Protein FoldIn 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