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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

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Comparison of T 1 and T 2

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Comparison of t 1 and t 2

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.


Comparison of t 1 and t 2

Problems with higher molecular weights and how to

overcome them

is the

linewidth

in Hz

at half peak

height


Comparison of t 1 and t 2

Pg 46 & 47 of Rattle


Comparison of t 1 and t 2

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


Comparison of t 1 and t 2

TROSY - Transverse Relaxation Optimised Spectroscopy

[Hz]

-50

50

ppm

Consider a 1H-15N HSQC peak

50

131

0

Decoupler switched on

-50

132

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).

90Hz

50

131

0

90Hz

-50

132

50

131

0

The pulse sequence selects just the sharp

component

-50

132

10.7

10.6

ppm


Comparison of t 1 and t 2

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.


Comparison of t 1 and t 2

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


Comparison of t 1 and t 2

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.


Comparison of t 1 and t 2

LFA-1

LFA-1


Comparison of t 1 and t 2

Developed small molecule inhibitors and test binding


Comparison of t 1 and t 2

Weak binding

mM to mM

see a migration of the peaks


Comparison of t 1 and t 2

It is straightforward to derive an expression for F([LTOT])

For the simplest case of a single ligand L, binding to a protein P


Comparison of t 1 and t 2

Total LFA-1 = 80M = [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

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


Comparison of t 1 and t 2

A more successful inhibitor- nM ‘tight’ binding.

See unbound and bound

populations


Comparison of t 1 and t 2

Solve NMR structure of complex…

Helix 7 is

prevented from

shifting


Comparison of t 1 and t 2

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.


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