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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.
Problems with higher molecular weights and how to
overcome them
1
v 
T2
v is the


linewidth
in Hz
at half peak
height
Pg 46 & 47 of Rattle
2H-labeling
for molecules greater than 25kDa
1H
-reduced relaxation (D/H ~ 1/6.5)
Dipole/Dipole
relaxation
-gives improved signal-to-noise
-better resolution
13C
H
H
D
H
H
H
N
D
D
D
H
N
TROSY - Transverse Relaxation Optimised Spectroscopy
[Hz]
-50
50
50
Consider a 1H-15N HSQC peak
131
0
-50
50
ppm
Decoupler switched on
132
90Hz
131
0
-50
132
50
131
0
-50
90Hz
132
10.7
10.6
ppm
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
component
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.
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
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
Inflammation (chronic) is responsible for asthma and arthritis.
LFA-1
LFA-1
Developed small molecule inhibitors and test binding
O-
O
O
N
S
A
B
C
N
N
D
N
O
Weak 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
Kd
PL  P + L
K d  dissociation constant for L dissociating from P
Define next the average number of ligand molecules bound to each protein, 
i.e.fraction of protein bound to ligand.
  conc. of L bound to P/total conc. of P =
[PL]
[P] + [PL]
[P][L]
[P] [L]
Kd
[L]
Since [PL] =
, then  =

[P][L] K d [L]
Kd
[P] +
Kd
If we measure a chemical shift change going from the free form to the fully bound
form then we can know  . We also know the amount of ligand we have added, so a
suitable plot allows us to determine the K d .
Total LFA-1 = 80mM = [P]+[PL]
L132 1H shift
Total ligand 20
m
50
100
150
200 400
NH of
L132
7.487 7.595 7.720 7.796 7.843 7.921

0.087 0.195

0.145 0.325
Bound Ligand
Free Ligand
11.6
8.4
26.0
24.0
0.320 0.396 0.443
0.521
100% bound
1H 8.0ppm
Unbound
1H 7.4ppm
0.534 0.660 0.738 0.869
42.7
57.3
52.8
59.0
69.5
97.2
141.0
330.5
=
[PL]
 [PL] /80
[P] + [PL]
i.e 0.145 * 80  11.6  [PL]

1
Plot 1.
 vs free ligand concentration
0.9
0.8
0.7

0.6
0.5
0.4
0.3
0.2
0.1
0
0
Plot 2. Better!
200
300
400
500
[L] (mM)
[L]
Kd  [L]
to give the equation of a straight line,

1



[L] K d K d
A plot of  /[Free Ligand] gives a slope
1
of ,here K d  50mM
Kd
0.018
0.016
/[Free Ligand]  M-
Rearrange  =
100
0.014
0.012
0.01
Series1
0.008
0.006
0.004
0.002
0
0
0.2
0.4
0.6

0.8
1
A more successful inhibitor- nM ‘tight’ binding.
See unbound and bound
populations
Solve NMR structure of complex…
Helix 7 is
prevented from
shifting
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.