OFF-RESONANCE ROTATING-FRAME NUCLEAR OVERHAUSER EFFECT SPECTROSCOPY

The incorporation of off-resonance spin-lock effects into the ROESY experiment enables the evaluation of nuclear motion and internuclear distances in macromolecules by employing the functional dependence of the cross-peak intensity on off-resonance spin-lock frequency. The salient difference between the off-resonance ROESY and the ROESY experiments is that the on-resonance spin lock was replaced by an off-resonance spin lock applied at frequency nu(off) and at field strength B-2 delivered in the form of a shifted laminar pulse. Variation of nu(off) at constant B-2 creates a suite of two-dimensional spectra with frequency-dependent dispersion behavior in the cross-peak intensity established by the interacting spin-1/2 nuclei. Simulations are presented describing the functional dependence of cross-peak intensity dispersion behavior on macromolecular parameters. This experiment, designated O-ROESY, was implemented in compensated form and yields an amplitude-corrected cross-peak intensity dispersion curve with a smoothly increasing NOESY contribution, at the expense of ROESY, as the off-resonance spin-lock frequency is increased. Application of the O-ROESY experiment was made to lysozyme, demonstrating the feasibility of obtaining the correlation time of specific internuclear vectors, the internuclear vector order parameter, the correlation time of overall reorientational motion, and the relative internuclear distance, from an analysis of O-ROESY cross-peak intensity dispersion curve behavior for several C-alpha proton pairs in the stable beta-strand region of the protein. (C) 1994 Academic Press.