title: Large multiple transmembrane domain fragments of a G protein-coupled receptor: biosynthesis, purification, and biophysical studies.
abstract: To conduct biophysical analyses on large domains of GPCRs, multimilligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of four transmembrane and five transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G(31) -A(198) ] (4TMN), four C-terminal Ste2p[T(155) -L(340) ] (4TMC), or five C-terminal Ste2p[I(120) -L(340) ] (5TMC) transmembrane segments of Ste2p. 4TMN was expressed as a fusion protein using a modified pMMHa vector in L-arabinose-induced Escherichia coli BL21-AI, and cleaved with cyanogen bromide. 4TMC and 5TMC were obtained by direct expression using a pET21a vector in IPTG-induced E. coli BL21(DE3) cells. 4TMC and 5TMC were biosynthesized on a preparative scale, isolated in multimilligram amounts, characterized by MS and investigated by biophysical methods. CD spectroscopy indicated the expected highly α-helical content for 4TMC and 5TMC in membrane mimetic environments. Tryptophan fluorescence showed that 5TMC integrated into the nonpolar region of 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) micelles. HSQC-TROSY investigations revealed that [(15) N]-labeled 5TMC in 50% trifluoroethanol-d(2) /H(2) O/0.05%-trifluoroacetic acid was stable enough to conduct long multidimensional NMR measurements. The entire Ste2p GPCR was not readily reconstituted from the first two and last five or first three and last four transmembrane domains.
authors.affiliation: Department of Chemistry, College of Staten Island, The City University of New York, Staten Island, NY 10314, USA.
authors.forename: Zhanna
authors.initials: Z
authors.lastname: Potetinova
authors.forename: Subramanyam
authors.initials: S
authors.lastname: Tantry
authors.forename: Leah S
authors.initials: LS
authors.lastname: Cohen
authors.forename: Katrina E
authors.initials: KE
authors.lastname: Caroccia
authors.forename: Boris
authors.initials: B
authors.lastname: Arshava
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; D032701:Biomimetics; D055592:Biophysical Phenomena; D002462:Cell Membrane / Q000737:chemistry; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D005822:Genetic Vectors / Q000737:chemistry; D002479:Inclusion Bodies / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids / Q000737:chemistry; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D051964:Receptors, Mating Factor / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D011993:Recombinant Fusion Proteins; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014364:Tryptophan; D003488:Cyanogen Bromide