Abstract
Two distinct time scales prevail in the study of conformational exchange in general and specifically in deriving the protein-bound conformations of ligands by two-dimensional transferred-NOE experiments. Fast-exchange compared to the chemical-shift difference leads to a complete averaging of the free and bound resonances of the ligands, while fast exchange compared to the longitudinal relaxation rate 1/ T 1 and cross relaxation results in an average of the relaxation matrices in the free and bound states. Slower chemical exchange leads to incomplete averaging of the free and bound resonances, giving rise to a reduction of transferred NOEs compared to fast-exchange conditions, Theoretical analysis and simulation results indicated that, in the absence of information on the chemical-shift time scale, transferred NOEs may be quantitated only to an accuracy with average NOE intensities and transferred NOEs to the free ligands as upper and lower bounds, respectively. Regardless of the exchange conditions, NOE intensities can be approximated by the NOEs transferred to the free ligands when a large excess of the free ligands (α b < 0.1) is used to amplify the NOE transfers between the bound protons, These results suggest that two-dimensional transferred-NOE experiments (transferred NOESY) can be applied in situations of intermediate and slow exchange as long as the fraction of bound ligands is small and the exchange rates are fast enough to transfer the cross-relaxation pathways in the protein-ligand complex to the easily detected resonances of the free ligands,
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