title: Slow internal dynamics in proteins: application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme.
abstract: Recently developed carbon transverse relaxation dispersion experiments (Skrynnikov, N. R.; et al. J. Am. Chem. Soc. 2001, 123, 4556-4566) were applied to the study of millisecond to microsecond time scale motions in a cavity mutant of T4 lysozyme (L99A) using methyl groups as probes of dynamics. Protein expressed in E. coli cells with (13)CH(3)-pyruvate as the sole carbon source contained high levels of (13)C enrichment at a total of 80 Val gamma, Leu delta, Ile gamma (2), Ala beta, and Met epsilon methyl positions with little extraneous incorporation. Data for 72 methyl groups were available for analysis. Dispersion profiles with large amplitudes were measured for many of these residues and were well fit to a two-state exchange model. The interconversion rates and populations of the states, obtained from fitting relaxation dispersion profiles of each individual probe, were remarkably homogeneous and data for nearly all methyl groups in the protein could be collectively fit to a single cooperative conformational transition. The present study demonstrates the general applicability of methyl relaxation dispersion measurements for the investigation of millisecond time scale protein motions at a large number of side-chain positions. Potential artifacts associated with the experiments are described and methods to minimize their effects presented. These experiments should be particularly well suited for probing dynamics in high molecular weight systems due to the favorable NMR spectroscopic properties of methyl groups.
authors.affiliation: Protein Engineering Network Centres of Excellence and Department of Medical Genetics, University of Toronto, Toronto M5S 1A8, Canada.
authors.forename: Frans A A
authors.initials: FA
authors.lastname: Mulder
authors.forename: Bin
authors.initials: B
authors.lastname: Hon
authors.forename: Anthony
authors.initials: A
authors.lastname: Mittermaier
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Dahlquist
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D002247:Carbon Isotopes; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D002247:Carbon Isotopes; D014764:Viral Proteins; D009113:Muramidase