Probing slow time scale dynamics at methyl-containing side chains in proteins by relaxation dispersion NMR measurements: Application to methionine residues in a cavity mutant of T4 lysozyme

A relaxation dispersion-based NMR experiment is presented for the measureme nt and quantitation of mus-ms dynamic processes at methyl side-chain positi ons in proteins. The experiment measures the exchange contribution to the C -13 line widths of methyl groups using a constant-time CPMG scheme. The eff ects of cross-correlated spin relaxation between dipole-dipole and dipole-C SA interactions as well as the effects of scalar coupling responsible for m ixing of magnetization modes during the course of the experiment have been investigated in detail both theoretically and through simulations. It is sh own that the complex relaxation properties of the methyl spin system do not complicate extraction of accurate exchange parameters as long as care is t aken to ensure that appropriate magnetization modes are interchanged in the middle of the constant-time CPMG period. An application involving the meas urement of relaxation dispersion profiles of methionine residues in a Leu99 Ala substitution of T4 lysozyme is presented. All of the methionine residue s are sensitive to an exchange event with a rate on the order of 1200 s(-1) at 20 degreesC that may be linked to a process in which hydrophobic ligand s are able to rapidly bind to the cavity that is present in this mutant.