2-DIMENSIONAL TRANSFERRED NUCLEAR-OVERHAUSER-EFFECTS WITH INCOMPLETE AVERAGING OF FREE-LIGAND AND BOUND-LIGAND RESONANCES

Two distinct time scales prevail in the study of conformational exchan ge in general and specifically in deriving the protein-bound conformat ions of ligands by two-dimensional transferred-NOE experiments. Fast-e xchange 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 fre e and bound states. Slower chemical exchange leads to incomplete avera ging of the free and bound resonances, giving rise to a reduction of t ransferred NOEs compared to fast-exchange conditions, Theoretical anal ysis and simulation results indicated that, in the absence of informat ion on the chemical-shift time scale, transferred NOEs may be quantita ted only to an accuracy with average NOE intensities and transferred N OEs to the free ligands as upper and lower bounds, respectively. Regar dless 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 (alpha(b) < 0.1) is used to amplify the NOE transfers be tween the bound protons, These results suggest that two-dimensional tr ansferred-NOE experiments (transferred NOESY) can be applied in situat ions of intermediate and slow exchange as long as the fraction of boun d ligands is small and the exchange rates are fast enough to transfer the cross-relaxation pathways in the protein-ligand complex to the eas ily detected resonances of the free ligands, (C) 1995 Academic Press, Inc.