Date of Award
1995
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
The primary focus of our investigations were centered around understanding the biochemical basis of the early events involved in signal transduction. Our work has involved two novel systems: (1) sphingolipid metabolites and (2) a synthetic peptide representing the transmembrane region of the human epidermal growth factor receptor (hEGFR).;Sphingolipid metabolites have emerged as physical modulators of many signalling pathways, including cell growth regulation, and signal transduction. N,N-({dollar}\sp2{dollar}H{dollar}\sb6{dollar}) -dimethylsphingosine and ({dollar}\sp2{dollar}H{dollar}\sb4{dollar}) -lysogalactosyl ceramide were produced by partial synthesis and studied via wideline {dollar}\sp2{dollar}H NMR, as a function of pH. Spectra of ({dollar}\sp2{dollar}H{dollar}\sb6{dollar}) -dimethylsphingosine identified that the molecule's orientation and behaviour are largely unaffected over the pH range studied. ({dollar}\sp2{dollar}H{dollar}\sb4{dollar}) -lysogalactosyl ceramide also demonstrated little variance over the pH range studied at the level of C{dollar}\sb4{dollar}, C{dollar}\sb5{dollar} of the sphingosine backbone, however the carbohydrate headgroup was seen to be orientationally altered.;The EGFR is an important example of the structural features associated with many receptor tyrosine kinases. A 34 amino acid peptide (hEGFR) representative of the transmembrane portion of the EGFR was synthesized possessing three selectively deuterated amino acid analogues (Ala{dollar}\sb{lcub}623{rcub}{dollar}, Met{dollar}\sb{lcub}644{rcub}{dollar}, Val{dollar}\sb{lcub}650{rcub}{dollar}), permitting investigation of its behaviour within a membrane environment via {dollar}\sp2{dollar}H NMR. This same peptide was structurally characterized by employing high resolution {dollar}\sp1{dollar}H NMR and circular dichroism spectroscopies.;The {dollar}\sp2{dollar}H NMR studies facilitated for the first time the identification of spectral features attributed to three spatially separated peptide/membrane environments. These deuteromethyl groups suggested that at elevated temperatures in fluid membranes the peptide behaves as a monomer. However, in the presence of cholesterol at temperatures below 40{dollar}\sp\circ{dollar}C the peptide demonstrated slowed axial rotation which is believed to be associated with increased oligomerisation.;Through the use of high resolution {dollar}\sp1{dollar}H NMR and circular dichroism spectroscopies, we have identified the presence of an {dollar}\alpha{dollar}-helix in the hEGFR peptide. Our {dollar}\sp1{dollar}H NMR study in aqueous TFE demonstrated that this helical segment commences at Met{dollar}\sb{lcub}626{rcub}{dollar}, and extends to Arg{dollar}\sb{lcub}647{rcub}{dollar}. The CD studies confirm the presence of a helical segment but suggest it is 17-19 residues in length.
Recommended Citation
Rigy, Alan Cavell, "High Resolution And Wideline Nmr Studies Of Membrane Associated Molecules Involved In Signal Transduction" (1995). Digitized Theses. 2573.
https://ir.lib.uwo.ca/digitizedtheses/2573