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