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 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.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/32836 |
| Date | 31 August 2012 |
| Creators | Tulumello, David |
| Contributors | Deber, Charles M. |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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