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Large multiple transmembrane domain fragments of a G protein‐coupled receptor: Biosynthesis, purification, and biophysical studies
Author(s) -
Potetinova Zhanna,
Tantry Subramanyam,
Cohen Leah S.,
Caroccia Katrina E.,
Arshava Boris,
Becker Jeffrey M.,
Naider Fred
Publication year - 2012
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.22122
Subject(s) - chemistry , transmembrane domain , transmembrane protein , g protein coupled receptor , biochemistry , stereochemistry , biophysics , membrane , receptor , biology
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. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 485–500, 2012.