| Title: | "Large multiple transmembrane domain fragments of a G protein-coupled receptor: biosynthesis, purification, and biophysical studies" |
| Author(s): | Potetinova Z; Tantry S; Cohen LS; Caroccia KE; Arshava B; Becker JM; Naider F; |
| Address: | "Department of Chemistry, College of Staten Island, The City University of New York, Staten Island, NY 10314, USA" |
| ISSN/ISBN: | 0006-3525 (Print) 1097-0282 (Electronic) 0006-3525 (Linking) |
| 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 alpha-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 alpha-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" |
| Keywords: | "Amino Acid Sequence Biomimetics Biophysical Phenomena Cell Membrane/chemistry Cyanogen Bromide/chemistry Electrophoresis, Polyacrylamide Gel Escherichia coli/chemistry Genetic Vectors/chemistry Inclusion Bodies/chemistry Magnetic Resonance Spectroscopy Mo;" |
| Notes: | "MedlinePotetinova, Zhanna Tantry, Subramanyam Cohen, Leah S Caroccia, Katrina E Arshava, Boris Becker, Jeffrey M Naider, Fred eng R01 GM022087/GM/NIGMS NIH HHS/ R56 GM022087/GM/NIGMS NIH HHS/ GM22087/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural 2012/12/04 Biopolymers. 2012; 98(5):485-500. doi: 10.1002/bip.22122" |
