title: Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor.
abstract: Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p. Ste2p TM1–TM3 (G31–R161) was expressed as a TrpΔLE fusion protein in Escherichia coli. The expressed protein was subject to CNBr cleavage to remove the fusion tag and TM1–TM3 was purified by reverse-phased HPLC. The cleavage product was isolated in yields of up to 20 mg per liter of culture in both unlabeled and uniformly [15N]-labeled and [15N, 13C, 2H]-labeled forms. The secondary structure of TM1–TM3 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and lysomyristoylphosphatidylglycerol (LMPG) detergent micelles by circular dichroism. Preliminary HSQC analysis in 50% TFE:water and LMPG micelles prepared in sodium phosphate and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffers revealed that this fragment is suitable for structural analysis by nuclear magnetic resonance (NMR). Complete backbone assignments and a detailed localization of the secondary structural elements of TM1–TM3 in 50% TFE:water have been achieved.
authors.affiliation: Department of Chemistry, The College of Staten Island, City University of New York, Staten Island, NY 10314, USA.
authors.forename: Katrina E
authors.initials: KE
authors.lastname: Caroccia
authors.forename: Racha
authors.initials: R
authors.lastname: Estephan
authors.forename: Leah S
authors.initials: LS
authors.lastname: Cohen
authors.forename: Boris
authors.initials: B
authors.lastname: Arshava
authors.forename: Melinda
authors.initials: M
authors.lastname: Hauser
authors.forename: Oliver
authors.initials: O
authors.lastname: Zerbe
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Becker
authors.forename: Fred
authors.initials: F
authors.lastname: Naider
mesh_terms: D000595:Amino Acid Sequence; D001703:Biophysics; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled