Structural characterization of the N-terminal region of the Saccharomyces cerevisiae G-protein coupled receptor, Ste2p
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G-protein coupled receptors (GPCRs) form a superfamily of cell surface receptors with in excess of 2000 genes identified across taxa (Pierce et al., 2002). They are integral membrane proteins that are comprised of seven hydrophobic helical segments which form a transmembrane spanning bundle. Ste2p and Ste3p are Saccharomyces cerevisiae GPCRs that are the α-factor and a-factor pheromone receptors, respectively (Bardwell, 2005). Ste2p in particular has served as an excellent model for studying the mechanisms of action of GPCRs. Recent results suggest that the extracellular N-terminus of the Ste2p receptor is involved in modulating cell wall degradation and membrane juxtaposition during yeast mating potentially by mediating an intercellular interaction with Ste3p (Shi et al., 2009a). The goals of this project were to obtain purified mg quantities of a soluble version of a portion of the N-terminus of Ste2p and to acquire structural information about this region by performing biophysical analysis on the soluble N-terminal Ste2p fragments. Initially, a synthetically produced KKK-Ste2p(14-43)-KKK peptide yielded circular dichroism (CD) spectra that indicated peptide secondary structure similar to what has been predicted in silico. Preliminary nuclear magnetic resonance (NMR) experiments with this peptide were also promising, yielding a correlation spectroscopy (COSY) spectrum that allowed for limited amino acid assignments. A recombinant version of the N-terminus of Ste2p, including residues 2-48 with terminal lysine residues, was expressed as a fusion protein in E. coli and the SK peptide was liberated by cyanogen bromide cleavage. Dynamic light scattering (DLS) analyses of the SK peptide showed it was aggregated when dissolved in water, but soluble in a trifluoroethanolamine/water (TFE/H2O) mixture. CD analysis of the SK peptide in TFE/H2O indicated that it contained more β-strand and less α-helix than the KKK-Ste2p(14-43)-KKK peptide. NMR analysis was performed on both an unlabelled and 15N-labelled SK peptide, yielding unusable spectra with very broad bands, most likely arising from aggregation at high concentrations. In conclusion, a high yield recombinant expression and purification system has been developed, yielding a Ste2p N-terminal peptide fragment that demonstrates some expected structural features. High resolution structural information may be obtained upon further optimization of the solvent system.
DegreeMaster of Science (M.Sc.)
SupervisorLoewen, Michele C.
CommitteeMoore, Stan; Dmitriev, Oleg; Roesler, Bill; Campanucci, Veronica
Copyright DateJuly 2011
G-protein coupled receptors
Nuclear magnetic resonance