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Title:Examining Molecular Interactions of Proteins by Isotopic Labeling, Sample Formulation and Solid-State NMR Spectroscopy
Author(s):Schmidt, Heather Lynn
Doctoral Committee Chair(s):Rienstra, Chad M.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Physical
Abstract:Advances in magic-angle spinning solid-state NMR (SSNMR) methods have led to several structural and functional studies of proteins. These methods can be extended to determine precise mechanistic details and molecular interactions of membrane or microcrystalline protein formulations. In this work, we developed techniques to examine molecular interactions in microcrystalline proteins and have begun efforts to identify mechanistic details of large membrane proteins by SSNMR. We first optimized expression, purification and SSNMR sample preparation methods of two large membrane proteins, E. coli cytochrome bo3 oxidase and A. thaliana cytochrome P450 monooxygenase 98A3. To obtain site-specific resolution in uniformly- 13C, 15N labeled samples of these proteins, hardware advances, new experimental techniques and increased dimensionality were combined. In addition, we developed methods to study electrostatic interactions of microcrystalline protein formulations. SSNMR chemical shift differences among microcrystalline formulations reported on salt bridges, intermolecular contacts and solvent interactions. For one microcrystalline form, acidic pKa values were determined, indicating their role in protein crystal stability. To apply these new techniques to large membrane proteins, we simplified chemical shift assignment procedures by using a pair-wise amino acid labeling technique. Using auxotroph E. coli strains, unambiguous amino acid pair assignments in the 144 kDa cytochrome bo3 oxidase was possible. This strategy will enable identification of key residues in the active site of this large enzyme, and will help elucidate mechanistic details.
Issue Date:2008
Description:180 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.
Other Identifier(s):(UMI)AAI3337917
Date Available in IDEALS:2014-12-17
Date Deposited:2008

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