Comparison of T 1 and T 2. rapid motion (small molecule non-viscous liquids), T 1 and T 2 are equal. Slow motion (large molecules, viscous liquids): T 2 is shorter than T 1. Problems with higher molecular weights and how to overcome them. is the linewidth in Hz at half peak
Comparison of T1 and T2
rapid motion (small molecule non-viscous liquids), T1 and T2 are equal
Slow motion (large molecules, viscous liquids): T2is shorter than T1.
Problems with higher molecular weights and how to
at half peak
Pg 46 & 47 of Rattle
2H-labeling for molecules greater than 25kDa
TROSY - Transverse Relaxation Optimised Spectroscopy
Consider a 1H-15N HSQC peak
Decoupler switched on
Decoupler switched off - 1J N-H 90 Hz
Each peak of the multiplet relaxes at a
different rate due to interference
between different relaxation mechanisms. This leads to broad (fast relaxing) and sharp components (slow relaxing).
The pulse sequence selects just the sharp
The NMR Bandshift and
binding site mapping
The 1H-15N HSQC spectrum is a
very powerful tool for rapid
monitoring of binding processes. If
the protein is 15N labeled then we
monitor chemical shift changes
caused by protein-protein interactions,
protein DNA interactions, protein-ligand
Examples right. Top, a 1H-15N HSQC of
an acyl carrier protein in the apo-form
(no fatty acid bound). In the lower
panel the effect of increasing fatty
acid chain length is monitored.
1. Screen for first ligand
3. Screen for second ligand
HSQC spectrum of a beta-lactamase in the absence (black)
and presence of inhibitor (red)
4. Optimise second ligand
5. Link ligands
Schematic of SAR by NMR
A case study - Leukocyte function associated protein-1 (LFA-1)
This protein is involved in tethering a leukocyte to a endothelium,
allowing migration through the tissue to a site of inflammation.
One domain of LFA-1, the I-domain is 181 amino acids and
undergoes a conformational change where helix 7 slides down the
protein, switching it into an active open form. This open form
is competent for cell surface binding.
If we can stop this switch, we may have an anti-inflammatory
Inflammation (chronic) is responsible for asthma and arthritis.
Developed small molecule inhibitors and test binding
mM to mM
see a migration of the peaks
It is straightforward to derive an expression for F([LTOT])
For the simplest case of a single ligand L, binding to a protein P
Total LFA-1 = 80M = [P]+[PL]
L132 1H shift
Total ligand 20 50 100 150 200 400
NH of 7.487 7.595 7.720 7.796 7.843 7.921
0.087 0.195 0.320 0.396 0.443 0.521
0.145 0.325 0.534 0.660 0.738 0.869
Bound Ligand 11.6 26.0 42.7 52.8 59.0 69.5
Free Ligand 8.4 24.0 57.3 97.2 141.0 330.5
A more successful inhibitor- nM ‘tight’ binding.
See unbound and bound
Solve NMR structure of complex…
Helix 7 is
NMR is a diverse tool with which we can study protein structure.
It gives us information in solution under ‘physiological’ conditions
2D and 3D techniques combined with modern assignment methods
have allowed proteins up to 40 kDa to be solved.
The power of NMR lies not just with its ability to solve structures
but also its ability to probe binding of ligands and partner proteins
in ‘real’ time.
Many aspects we have not had time to deal with. NMR reveals how
proteins move in solution - can see domains flexing with different
timescale motions. These often correlate with binding patches
on the protein.