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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
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
Put Residues In SequencePut Residues In Sequence
A B C D Z• • • • Intraresidue
Sequential
Medium-range(helices)
Tertiary Structure
Use only these to make sequential assignments
Sequential NOEs
-strand H(i-1) -> HN(i) -helix :H(i-1) -> HN(i)
NOE based Assignment
1515N edited N edited 11H-H-11H TOCSY/NOESYH TOCSY/NOESY
Large Scalar Couplings Large Scalar Couplings Less Less Sensitive to MW of the ProteinSensitive 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
Heteronuclear Assignments:Heteronuclear Assignments:Backbone ExperimentsBackbone Experiments
Names of scalar experiments based on atoms detected Acquired in ‘pairs’
Heteronuclear Assignments:Heteronuclear Assignments:Side Chain ExperimentsSide Chain Experiments
Determining Protein FoldIn Structure Calculations
1. Determine secondary structure•CSI directly from assignments•Medium-range NOEs
2. Add key long-range NOEs to fold
Identify more NOEs
In the course of refinement