Membrane proteins are responsible for a variety of key cellular functions including transport of essential substrates across the membrane, signal transduction, and maintenance of cellular morphology. However, given the size and high hydrophobicity of membrane proteins, along with demanding expression and solubilization protocols that often preclude biophysical studies, novel approaches must be devised for studies of their structure and function. This thesis addresses these issues through several sets of inter-related experiments. We first examine sequence motifs directing -helix packing, wherein the determinants of glycophorin A (GpA) dimerization were identified via TOXCAT assay and the evaluation of GpA-derived peptides. We found that (i) conservative mutations can have significant effects on the oligomerization of glycophorin A; and (ii) residues that introduce more efficiently packed structures that are poorly solvated by lipid leads to improved transmembrane segment dimerization. In a further study, we inquired into the criteria for selection of membrane-spanning -helices by cellular machinery through investigation of hydrophobic helical segments (termed -helices) that we identified in soluble proteins. We found that the number and location of charged residues in a given hydrophobic helix are related to their insertion propensity as membrane-spanning segments. When we applied this criterion to -helices in their intact protein structures, we successfully determined the extent of -helix mutations necessary to convert a soluble protein, in part, to a membrane-inserted protein. Finally, using a three-transmembrane segment construct from the cystic fibrosis transmembrane conductance regulator (CFTR), we performed experiments aimed at optimizing criteria for protein overexpression, including construct design, choice of expression system, growth media, and expression temperature. The overall findings are interpreted in terms of progress towards defining the fundamental characteristics of membrane-spanning -helices - from their primary amino acid sequence to the helix-helix interactions they display in the assembly of biologically-functional membrane protein structures.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31727 |
| Date | 05 January 2012 |
| Creators | Cunningham, Fiona |
| Contributors | Deber, Charles M. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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