Transcript Center for Structural Biology

NMR Scalar and Dipolar Coupling
Through
Space
Through
Bonds
Coupling of nuclei gives information on structure
Resonance Assignment
CH3-CH2-OH
OH
CH2 CH3
Which signal from which H atoms?
The key attribute: use the scalar and dipolar couplings to
match the set of signals with the molecular structure
Proteins Have Many Signals
1H
NMR Spectrum of Ubiquitin
~500 resonances
A large number of signals are overlapped
Protein NMR:
Overlapped Sub-Spectra
Each residue in the sequence gives rise to an
independent NMR sub-spectrum, which is much
simpler than the complete protein spectrum
Methods have been
developed to extract
each sub-spectrum
from the whole
Simplifies Strategy to Assign
Resonances in a Protein
1. Identify resonances for each amino
acid
T G L S
R G
S
2. Put amino acids in order (2 steps)
- Find next neighbors (R-G-S,T-L-G-S)
- Sequence-specific assignment
1
2
3
4
5
6
7
R-G-S-T-L-G-S
Homonuclear 1H Assignment Strategy
• Scalar coupling to identify resonances, dipolar
couplings to place in sequence
• Based on backbone NH
• Concept: build out from the backbone to
identify the side chain resonances
• 2nd dimension resolves overlaps, 3D rare
1H
1H
1H
COSY: One J coupling
H
A
N—C
H
B
H
N—C
H
C
H
H
N—C
R-COSY: Add A 2nd J Coupling
H
A
N—C—C
H
B
H H
N—C—C
H
C
H H
H
N—C—CH3
DR-COSY: Add A 3rd J Coupling
H
A
H H
N—C—C
H
H
B
H H
N—C—C
H
H
C
H
N—C—CH3
TOCSY: All Coupled Spins
H
A
H H
H
N—C—C—C—COOH
H H
H
B
H H
H
H
N—C—C—C—C—C—NH3
H H
H
C
H
H
N—C—CH3
H
H
Heteronuclear (1H,13C,15N) Strategy
• One bond at a time - all atoms (except O)
• Even handles backbone 15N1H overlaps
 disperse with backbone
C’CaHaCbHb…
• Works on bigger proteins because one
bond scalar couplings are larger