Biosynthesis, purification and biophysical analysis of domains of the Saccharomyces cerevisiae alpha-factor receptor

The α-factor receptor (Ste2p) is an essential protein for the sexual conjugation of the yeast Saccharomyces cerevisiae. It belongs to the family of G protein-coupled receptors, and it is believed to share a common structural motif with the other members of the family; seven transmembrane segments, that are α-helices within the membrane, connected by alternating extracellular and intracellular loops.; In the current thesis project, studies were performed on the M5I3M6 region that corresponds to transmembrane domains 5 and 6 and the connecting third intracellular loop to complement the biophysical studies carried out on single transmembrane regions and the intracellular and extracellular loops of Ste2p (Reddy et al., 1994; Arshava et al., 1998; Xie et al., 2000; Ding et al., 2001). Molecular biology techniques were used to express the M5I3M6 and M6 segments of Ste2p fused to a leader protein in E. coli. These polypeptides were isolated and purified in 10–100 mg quantities. The fusion proteins were characterized by gel electrophoresis, mass spectrometry and amino acid analysis. Their secondary structures were investigated in membrane mimetic environments including trifluoroethanol:water mixtures, SDS micelles, and lipid vesicles using circular dichroism. The structures of M5I3M6 and M6 linked to a solubilizing leader peptide were partial helices.; The CD studies initiated by Xie and others on single transmembrane domains of Ste2p were extended by applying nuclear magnetic resonance (NMR) spectroscopy to investigate the M6 region. NMR experiments were performed in CDCl ₃:CD₃OH:H₂O (4:4:1) to obtain a high resolution structure of the sixth transmembrane region. Different models were calculated for a wildtype peptide sequence and for an analog containing a Pro to Leu mutation that causes Ste2p to become constitutively active. The helical structure found in these membrane-spanning peptides is consistent with the findings published by Xie et al. (2000) and provides biophysical verification of hydropathy predictions on intact receptor.