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Title:Multidimensional Solid-State NMR Studies of Membrane Proteins
Author(s):Li, Ying
Doctoral Committee Chair(s):Rienstra, Chad M.
Department / Program:Center for Biophysics and Computational Biology
Discipline:Biophysics and Computational Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Chemistry, Physical
Biophysics, General
Abstract:Recent progress in solid-state NMR techniques presents unique opportunities to study the structure and dynamics of membrane proteins by using uniformly or extensively isotopically labeled samples. In this work, we first optimized the performance of shaped pulses in magic-angle spinning solid-state NMR experiments by characterizing their frequency profiles and refocusing efficiency under conditions commonly used for protein spectroscopy. We have also identified the correct rotor-synchronization condition, which allowed efficient refocusing of J-coupling in the indirect dimensions and led to significant improvement in spectral resolution. We then applied multidimensional magic-angle spinning NMR techniques to systems including the membrane protein DsbB, Nanodisc, a self-assembled lipid bilayer system, and Nanodisc-embedded human cytochrome P450 3A4. For DsbB, nearly complete 13C and 15N chemical shift assignment was achieved for the transmembrane region of the protein from 3D and 4D chemical shift correlation experiments. Nanodisc is a nanoscale lipid bilayer system that has been designed to facilitate the biochemical and biophysical studies of membrane proteins. In order to evaluate the potential of utilizing Nanodiscs for solid-state NMR study of membrane proteins, we first prepared samples of empty Nanodisc with the membrane scaffold protein isotopically labeled. We demonstrated that precipitated Nanodisc samples show narrow inhomogeneous linewidths, which are beneficial for further structural studies. We also showed that 2D data strongly support one of the two prevailing computational models of membrane scaffold protein, which further proves that the structural integrity of Nanodiscs was not affected by the precipitation protocol we employed. Based on these results, we initiated the study of human cytochrome P450 3A4 embedded into Nanodiscs. We showed that solid-state NMR samples of cytochrome P450 3A4 can be obtained with its structural integrity maintained by using essentially the same precipitation protocol. The initial NMR results confirmed the essential sample properties required for further studies. Overall, the work presented in this dissertation demonstrates the potential of using magic-angle spinning NMR technique to solve high-resolution structures of membrane proteins in a lipid environment that resembles the cell membrane.
Issue Date:2008
Description:196 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.
Other Identifier(s):(UMI)AAI3314838
Date Available in IDEALS:2014-12-17
Date Deposited:2008

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