PROTEIN INDIRECT RELAXATION EFFECTS IN EXCHANGE-TRANSFERRED NOESY BY A RATE-MATRIX ANALYSIS

The influence of relaxation by protein protons on ligand-ligand intensities measured by two-dimensional exchange-transferred nuclear Overhauser spectroscopy (ET-NOESY) is examined by simulation studies using a rate-matrix analysis. Evidence that effects from the protein protons can be significant is demonstrated by experimental data from the complex NADH · lactate dehydrogenase. Both protein and ligand protons are explicitly considered in a rate matrix comprising chemical-exchange and NMR relaxation rates. In the fast-exchange limit with respect to magnetic relaxation rates, the matrix equation simplifies to a symmetrical form, so that standard symmetric matrix diagonalization methods can be used to solve the equation. Computer simulation studies for a three-spin system show that, for certain geometries, protein protons have significant indirect effects on ligand-ligand cross-peak intensities which could lead to inaccurate estimates for the distances between ligand protons. The simulation studies also show that T1 of the free ligand influences the observed exchange-transferred NOE. Analysis of experimental ET-NOE data finds that protein relaxation effects are present; a comparison between expected ET-NOESY intensities based on crystallographic coordinates and those measured experimentally for NADH-lactate dehydrogenase indicates a significant indirect effect from Ser 137 Hα of the protein to the ligand-ligand cross peak H1′-H4′ of the ribose of the ribonicotinamide moiety. © 1993 Academic Press, Inc.