Transcript Use only these to make sequential assignments

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 Sequence
Use only these to make
sequential assignments
Tertiary Structure
Sequential
Intraresidue
A
B
C
Medium-range
(helices)
D
••••
Z
Sequential NOEs
b-strand Ha(i-1) -> HN(i)
a-helix :HN(i-1) -> HN(i)
NOE based Assignment
15N
edited
1H-1H TOCSY/NOESY
Large Scalar Couplings  Less
Sensitive 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:
Backbone Experiments
Names of scalar
experiments based
on atoms detected
Acquired in ‘pairs’
Heteronuclear Assignments:
Side Chain Experiments
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
Identify more NOEs
In the course of refinement