Type of Document	Dissertation
Author	Page, Richard C.
URN	etd-03312008-100356
Title	Structural Characterizations of Integral Membrane Proteins: The Nexus of Solution and Solid State NMR Spectroscopy
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Timothy Cross Committee Chair John Dorsey Committee Member Richard Bertram Committee Member Timothy Logan Committee Member
Keywords	Solid State NMR Membrane Proteins Structural Biology Biophysics Solution NMR
Date of Defense	2008-03-19
Availability	unrestricted
Abstract Membrane proteins account for 1/3 of the proteins encoded within known genomes and at least 60% of current drug targets. While the field of structural biology has achieved remarkable success in determining the three dimensional structures of water soluble, globular proteins, conventional techniques for soluble protein structure determination have seen limited success when applied to integral membrane proteins. Improving the methods used for membrane protein structure determination is crucial to increasing the number of known membrane protein structures and to improving our understanding of these vital proteins. Herein we focus on tackling the tough issues facing á-helical integral membrane protein structure determination: expression, purification, sample preparation, and methodologies for characterization by solution and solid state nuclear magnetic resonance (NMR) spectroscopy. Membrane protein expression, purification and sample preparation techniques are examined and applied to two integral membrane proteins, KdpC (Rv1031) and Rv1761c from Mycobacterium tuberculosis. Two distinct sample preparation methods capable of producing homogeneous and reproducible high quality samples are discussed for solution NMR studies of membrane proteins. The use of these sample preparation methods has allowed for the backbone structures of Rv1761c and the transmembrane domain of KdpC to be determined. These structures and the effects of detergent micelles upon integral membrane proteins are discussed, and a method is proposed for circumventing some of the undesirable attributes of detergent micelle samples. Improvements in the analysis of solid state NMR experiments in oriented lipid bilayer samples are proposed including a torsion angle mapping method for examining torsion angles directly from two dimensional solid state NMR polarization inversion of spin exchange at the magic angle (PISEMA) spectra. The torsion angle mapping analysis is discussed in terms of enhancing the utility of solid state NMR spectroscopy for characterizing integral membrane proteins in synthetic lipid bilayers.
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