Date of Award
Doctor of Philosophy (PhD)
Membrane proteins are an integral part of the structure of membranes providing the functional aspect of the membrane with respect to the transport of water soluble molecules, signal recognition and reaction cataylsis. Primary, secondary and tertiary structure of these types of proteins warrant investigation in order to better understand their individual function. Both DNA sequencing and standard protein sequencing can provide information on the protein's primary sequence. Protein sequencing can identify sites of post-translational peptide bond hydrolysis and translocation and aid in the elucidation of the secondary and tertiary structure. However, the hydrophobic nature of these membrane proteins necessitates the use of detergents or chaotropic agents to achieve the solubilization of the protein and even its peptide fragments under aqueous conditions; in the absence and sometimes even in the presence of these agents, membrane proteins aggregate and precipitate out of solution. Standard protein sequencing techniques are, therefore, hampered by this protein aggregation and insolubility, resulting in incomplete enzyme and chemical cleavage of the protein, general losses of both the protein and the peptide fragments and the overall poor isolation of hydrophobic peptide fragments from one another.
The focus of this research was on the development of a method by which membrane proteins could be chemically derivatized resulting in the formation of a stable product that was soluble in aqueous solutions, in the absence of detergents and chaotropic agents. The methodology consists of the following: 1. Solubilization of the membrane protein in anhydrous pyridine through the utilization of tetrabutylammonium ions which provide a hydrophobic counter ion to the carboxylate groups on the protein. 2. Complete derivatization of the amino add side chain amine and hydroxyl groups with di-trimethylsilylethyl trimesic anhydride, in the presence of the acylation catalyst, 4-pyrrolidinopyridine. 3. Regeneration of the carboxyl groups on the trimesic acid moeity through the selective removal of the trimethylsilylethyl ester groups by tetrabutylammonium fluoride. 4. Isolation of the trimesylated protein by column chromatography or dialysis. Apobacteriorhodopsin was used as the model membrane protein and after this procedure, the trimesylated protein was found to be completely soluble in simple aqueous solutions. This water soluble derivative was shown to chromatograph on Sephadex type columns that were eluted with buffers containing low salt concentrations. It was further shown to be a suitable substrate for the enzyme pyroglutamate aminopeptidase resulting in the quantitative removal of the N-terminal pyroglutamate residue; the native protein is a poor substrate due to its insolubility, under these aqueous conditions.
This method of trimesylation is applicable to all proteins, both membranous and water soluble, generating a trimesylated protein derivative that is completely water soluble. This chemical modification incorporates two carboxyl groups at each site of modification, Lys, Tyr, Ser and Thr side chains; due to the frequency of the hydroxyl amino acid residues even in hydrophobic domains, this modification would also enable peptide fragments to be water soluble.
Morton, Robert Christopher, "Acylation of Membrane Proteins to Enhance Their Water Solubility" (1991). Open Access Dissertations and Theses. Paper 3613.