SOME NMR EXPERIMENTS AND A STRUCTURE DETERMINATION EMPLOYING A (N-15,H-2) ENRICHED PROTEIN

We present the results of studies of an aqueous sample of a highly {N- 15,H-2} enriched protein, the SH3 domain from Fyn. Measurements of H-1 relaxation and interactions between H2O solvent and exchangeable prot ons are given, as well as a method for increasing the effective longit udinal relaxation of solvent exchangeable proton resonances. The long- range isotope shifts are measured, for H-1 and N-15, which arise due t o perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time s eries of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this H-1 dilute sample, r elatively long mixing times (up to 1.2 s) can be used to detect specif ic dipolar interactions between amide protons up to about 7 Angstrom a part. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus id entified in a series of 3D HSQC-NOESY-HSQC experiments. These data, al one and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compa red to the available X-ray and NMR structures of the Fyn SH3 domain.