Comparison of T 1 and T 2

1. 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.

2. Problems with higher molecular weights and how to overcome them is the linewidth in Hz at half peak height

3. Pg 46 & 47 of Rattle

5. 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

7. 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

8. 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.

9. 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

10. 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.

11. LFA-1 LFA-1

13. Developed small molecule inhibitors and test binding

14. Weak binding mM to mM see a migration of the peaks

16. It is straightforward to derive an expression for F([LTOT]) For the simplest case of a single ligand L, binding to a protein P

17. 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

19. A more successful inhibitor- nM ‘tight’ binding. See unbound and bound populations

20. Solve NMR structure of complex… Helix 7 is prevented from shifting

21. 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.