Intermolecular relaxation has little effect on intra-peptide exchange-transferred NOE intensities.

Abstract

Exchange-transferred nuclear Overhauser enhancement (etNOE) provides a useful method for determining the 3dimensional structure of a ligand bound to a high-molecular-weight complex. Some concern about the accuracy of such structures has arisen because indirect relaxation can occur between the ligand and macromolecule. Such indirect relaxation, or spin diffusion, would lead to errors in interproton distances used as restraints in structure determination. We address this concern by assessing the extent of intermolecular spin diffusion in nineteen peptide-protein complexes of known structure. Transferred NOE intensities were simulated with the program CORONA (Calculated OR Observed NOESY Analysis) using the rate-matrix approach to include contributions from indirect relaxation between protein-ligand and intraligand proton pairs. Intermolecular spin diffusion contributions were determined by comparing intensities calculated with protonated protein to those calculated with fully deuterated protein. The differences were found to be insignificant overall, and to diminish at short mixing times and high mole ratios of peptide to protein. Spin diffusion between the peptide ligand and the protein contributes less to the etNOE intensities and alters fewer cross peaks than the well-studied intramolecular spin diffusion effects. Errors in intraligand interproton distances due to intermolecular relaxation effects were small on average and can be accounted for with the restraint functions commonly used in NMR structure determination methods. In addition, a rate-matrix approach to calculate distances from etNOESY intensities using a volume matrix comprising only intraligand intensities was found to give accurate values. Based on these results, we conclude that structures determined from etNOESY data are no less accurate due to spin diffusion than structures determined from conventional NOE intensities.