NMR methods for studying the dynamics of proteins in solution.
Published: 2004
Language: eng
Number of pages: 203
Description: Nuclear magnetic resonance (NMR) can characterize protein internal motions on timescales ranging from picoseconds to days with atomic resolution. This thesis describes the measurement of physical parameters critical for the accurate interpretation of NMR dynamics results, the development of spectroscopic and data analysis techniques, and applications to the study of protein function and stability.New NMR pulse schemes allowing the measurement of 13C beta-1Hbeta dipolar couplings in randomly fractionally deuterated proteins are presented. Data have been obtained using two alignment media and are used to fit chi1 torsion angles, identify residues undergoing rotameric jumps and determine rotamer populations.15N and 2H dynamics data for eight proteins have been compiled and compared to the corresponding molecular structures. Surprisingly, the correlations between side-chain flexibility and solvent accessibility and packing density are quite weak. A significantly stronger correlation is observed for evolutionary sequence conservation, such that residues that are highly preferred at a given position tend to be less mobile.15N and 13C relaxation dispersion experiments have been performed on a cavity mutant of T4 lysozyme in order to characterize a weakly populated excited state. The relative entropy and enthalpy of this state as well the activation energy of the transition have been calculated from the temperature dependence of populations and exchange rates.Spin relaxation experiments have been used to characterize the response of internal dynamics to hydrophobic core mutations in two protein domains. Extensive simulations have been performed and three-bond scalar coupling measurements recorded in order to discriminate between changes in the magnitude and changes in the timescale of side-chain dynamics.Information about side-chain motions can be extracted from deuterium relaxation rates measured for randomly fractionally deuterated proteins. Correct interpretation of the data in terms of conformational flexibility requires accurate knowledge of the quadrupolar coupling constant. Quadrupolar coupling data from a protein sample weakly aligned in solution show that the constant is uniform for methyl deuterons and give a value in good agreement with prior results. A similar liquid crystal approach indicates that negligible differences exist among the geometries of CH3, CH2D and CHD 2 methyl isotopomers.
Notes: Adviser:  Lewis E. Kay.

Thesis (Ph.D.)--University of Toronto, 2003 (copyright 2004).

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2401.