Resonance Assignment NMR analysis of proteins Sequential resonance assignment strategies Practical...
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Transcript of Resonance Assignment NMR analysis of proteins Sequential resonance assignment strategies Practical...
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