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Detergents As Membrane-Mimetic Media for Structural Characterization of Membrane Proteins

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Detergents As Membrane-Mimetic Media for Structural Characterization of Membrane Proteins Detergents as membrane-mimetic media for structural characterization of membrane proteins. by David Vincent Tulumello A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Biochemistry University of Toronto © Copyright by David Vincent Tulumello 2012 Detergents as membrane-mimetic media for structural characterization of membrane proteins. David Vincent Tulumello Doctor of Philosophy Department of Biochemistry University of Toronto 2012 Abstract Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent ii alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two- transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure. iii Acknowledgments It is my pleasure to thank all those who have made completing this thesis possible. I am extremely grateful to my supervisor Dr. Charles Deber. He has been a great mentor and teacher, and has always displayed the utmost devotion to my professional growth and academic training. It has been a privilege to model myself as a scientist based on his admirable example. Working and learning alongside all of the current and former members of the Deber lab has been a pleasure. They have always been willing and generous in sharing both their time and expertise. Through their kindness and friendship, my colleagues have made spending time in the lab an enjoyable process. I am also thankful to the many scientists who have provided guidance throughout my development as a researcher. These include members of my supervisory committee, Dr. Alan Davidson and Dr. Igor Stagljar, collaborators from the University of Toronto Mississauga Campus, especially Dr. Scott Prosser, and former supervisors from my undergraduate studies at McMaster University, Dr. Adam Hitchcock and Dr. Graham McGibbon. I would like to express deep gratitude to my parents, Mary and Peter. They were the first to encourage my love of science by fostering my curiosity at a young age. Instead of simply answering my many questions about how the world works, they chose to show me. In many ways, I consider them to be my very first scientific advisors. Finally, I have been truly blessed for having the loving support of my wife, Amanda. She has continuously provided me with the strength and motivation to persevere through the hard times, and has helped me pause to celebrate the successes. She has been by my side during every phase of my graduate career, and without her, this thesis would not have been possible. iv Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Tables .................................................................................................................................. x List of Figures ................................................................................................................................ xi List of Appendices ....................................................................................................................... xiii List of Abbreviations ................................................................................................................... xiv Chapter 1. Introduction. ............................................................................................................ 1 1.1 Membrane proteins. ............................................................................................................ 2 1.2 Transmembrane protein structure. ...................................................................................... 3 1.2.1 Structure and function of β-barrel membrane proteins. .......................................... 4 1.2.2 Structural and function of α-helical membrane proteins. ....................................... 7 1.2.3 Properties of α-helical transmembrane segments. .................................................. 9 1.3 Transmembrane protein folding. ....................................................................................... 10 1.3.1 Bilayer insertion. ................................................................................................... 10 1.3.2 Helix – helix association. ...................................................................................... 11 1.3.3 Helix – lipid interactions. ...................................................................................... 13 1.3.4 Additional features of transmembrane domain assembly. .................................... 14 1.4 Interaction of transmembrane proteins with detergents. ................................................... 15 1.4.1 Detergent properties. ............................................................................................. 16 1.4.2 Solubilization versus stabilization: the role of detergents in membrane protein characterization. .................................................................................................... 17 1.4.3 Overview of commonly used detergents for membrane protein characterization. .................................................................................................... 18 1.5 Characterization of transmembrane proteins in detergents. .............................................. 22 1.5.1 General considerations. ......................................................................................... 22 v 1.5.2 Detergent micelle insertion and secondary structure. ........................................... 24 1.5.3 Transmembrane protein tertiary and quaternary structure analysis. ..................... 26 1.5.4 High resolution structure determination. .............................................................. 29 1.6 Thesis hypothesis and outline. .......................................................................................... 31 Chapter 2. SDS micelles as a membrane-mimetic environment for transmembrane segments………. ...................................................................................................................... 33 2.1 Introduction. ...................................................................................................................... 34 2.2 Materials and methods. ..................................................................................................... 35 2.2.1 Transmembrane segment prediction. .................................................................... 35 2.2.2 Peptide synthesis and purification. ....................................................................... 35 2.2.3 TOXCAT assays. .................................................................................................. 36 2.2.4 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. ................................................................................................................. 36 2.2.5 Circular dichroism spectroscopy. .......................................................................... 37 2.2.6 Tryptophan fluorescence measurements. .............................................................. 37 2.2.7 Förster resonance energy transfer (FRET). ........................................................... 38 2.3 Results. .............................................................................................................................. 38 2.3.1 Peptide design and TM insertion prediction. ........................................................ 38 2.3.2 Oligomerization
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