Comparison of T1 and T2

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Comparison of T1 and T2
rapid motion (small molecule non-viscous
liquids), T1 and T2 are equal
Slow motion (large molecules, viscous liquids)
T2 is shorter than T1.
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Problems with higher molecular weights and how
to overcome them
is the linewidth in Hz at half peak height
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Pg 46 47 of Rattle
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2H-labeling for molecules greater than 25kDa
1H

• reduced relaxation (?D/?H 1/6.5)
• gives improved signal-to-noise
• better resolution

Dipole/Dipole relaxation
13C
D
D
H
H
D
H
D
H
H
H
N
N
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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 interactions. 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.
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1. Screen for first ligand
2. Optimise 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
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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 mechanism Inflammat
ion (chronic) is responsible for asthma and
arthritis.
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LFA-1
LFA-1
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Developed small molecule inhibitors and test
binding
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Weak binding mM to mM see a migration of the peaks
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It is straightforward to derive an expression for
F(LTOT) For the simplest case of a single
ligand L, binding to a protein P
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Total LFA-1 80?M PPL
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 L132 ?? 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
100 bound 1H 8.0ppm
Unbound 1H 7.4ppm
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A more successful inhibitor- nM tight binding.
See unbound and bound populations
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Solve NMR structure of complex
Helix 7 is prevented from shifting
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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.