The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339) containing the third extracellular loop, the seventh transmembrane domain, and 40 residues of the cytosolic tail (E3-M7-24-T40) was biosynthesized fused to a carrier protein. The multidomain fusion protein (designated M7FP) was purified to near homogeneity as judged by HPLC and characterized by mass spectrometry. In minimal medium, 30-40 mg of M7FP were obtained per liter of culture. The 73-residue peptide was released from its carrier by CNBr and obtained in wild-type, (15)N, and (13)C/(15)N forms. The E3-M7-24-T40 peptide integrated into 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] and dodecylphosphocholine micelles at concentrations (200-500 microM) suitable for NMR investigations. HSQC experiments performed in organic solvents and detergent micelles on (15)N-labeled E3-M7-24-T40 showed a clear dispersion of the nitrogen-amide proton correlation cross-peaks indicative of a pure, uniformly labeled molecule that assumed a partially ordered structure. NOE connectivities, chemical shift indices, J-coupling analysis, and structural modeling suggested that in trifluoroethanol/water (1:1) helical subdomains existed in both the transmembrane and cytoslic tail of the multidomain peptide. Similar conclusions were reached in chloroform/methanol/water (4:4:1). As the cytosolic tail participates in down-regulation of Ste2p, the helical regions in the Ste2p tail may play a role in protein-protein interactions involved in endocytosis.
Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments.
Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments.
Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments.
Biochim Biophys Acta. 2011 Jul 23;
Authors: Cohen LS, Arshava B, Neumoin A, Becker JM, Güntert P, Zerbe O, Naider F
Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a...
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07-28-2011 10:51 AM
Mutations in the Saccharomyces cerevisiae succinate dehydrogenase result in distinct metabolic phenotypes revealed through (1)H NMR-based metabolic footprinting.
Mutations in the Saccharomyces cerevisiae succinate dehydrogenase result in distinct metabolic phenotypes revealed through (1)H NMR-based metabolic footprinting.
Mutations in the Saccharomyces cerevisiae succinate dehydrogenase result in distinct metabolic phenotypes revealed through (1)H NMR-based metabolic footprinting.
J Proteome Res. 2010 Dec 3;9(12):6729-39
Authors: Szeto SS, Reinke SN, Sykes BD, Lemire BD
Metabolomics is a powerful method of examining the intricate connections between mutations, metabolism, and disease. Metabolic...
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05-25-2011 07:01 PM
NMR solution structure of subunit E (fragment E(1-69)) of the Saccharomyces cerevisiae V (1)V (O) ATPase.
NMR solution structure of subunit E (fragment E(1-69)) of the Saccharomyces cerevisiae V (1)V (O) ATPase.
NMR solution structure of subunit E (fragment E(1-69)) of the Saccharomyces cerevisiae V (1)V (O) ATPase.
J Bioenerg Biomembr. 2011 Mar 12;
Authors: Rishikesan S, Thaker YR, Grüber G
The N-terminus of V-ATPase subunit E has been shown to associate with the subunits C, G and H, respectively. To understand the assembly of E with its neighboring subunits as well as its N-terminal structure, the N-terminal region, E(1-69), of the...
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03-15-2011 04:06 PM
[NMR paper] Solution NMR spectroscopy of the human vasopressin V2 receptor, a G protein-coupled r
Solution NMR spectroscopy of the human vasopressin V2 receptor, a G protein-coupled receptor.
Related Articles Solution NMR spectroscopy of the human vasopressin V2 receptor, a G protein-coupled receptor.
J Am Chem Soc. 2005 Jun 8;127(22):8010-1
Authors: Tian C, Breyer RM, Kim HJ, Karra MD, Friedman DB, Karpay A, Sanders CR
The seven-transmembrane-spanning G protein-coupled receptor (GPCR) superfamily plays many important roles in basic biology, human health, and human disease. Here, well-resolved solution NMR spectra are presented for a human...
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11-25-2010 08:21 PM
[NMR paper] The linker histone homolog Hho1p from Saccharomyces cerevisiae represents a winged he
The linker histone homolog Hho1p from Saccharomyces cerevisiae represents a winged helix-turn-helix fold as determined by NMR spectroscopy.
Related Articles The linker histone homolog Hho1p from Saccharomyces cerevisiae represents a winged helix-turn-helix fold as determined by NMR spectroscopy.
Nucleic Acids Res. 2003 Dec 15;31(24):7199-207
Authors: Ono K, Kusano O, Shimotakahara S, Shimizu M, Yamazaki T, Shindo H
Hho1p is assumed to serve as a linker histone in Saccharomyces cerevisiae and, notably, it possesses two putative globular...
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11-24-2010 09:16 PM
[NMR paper] Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)
Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)H, (13)C and (15)N chemical shift assignments by multidimensional NMR.
Related Articles Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)H, (13)C and (15)N chemical shift assignments by multidimensional NMR.
FEBS Lett. 2000 Sep 29;482(1-2):25-30
Authors: Szabo CM, Sanders LK, Le HC, Chien EY, Oldfield E
We have expressed -labeled Saccharomyces cerevisiae iso-1 cytochrome c C102T;K72A in Escherichia coli with a yield of 11...
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11-19-2010 08:29 PM
[NMR paper] NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a
NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.
Related Articles NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.
J Mol Biol. 1999 Aug 20;291(3):661-9
Authors: Evans SP, Bycroft M
In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small...
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11-18-2010 08:31 PM
[NMR paper] Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the Sac
Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the Saccharomyces cerevisiae tridecapeptide pheromone receptor.
Related Articles Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the Saccharomyces cerevisiae tridecapeptide pheromone receptor.
Biopolymers. 1998 Nov;46(6):343-57
Authors: Arshava B, Liu SF, Jiang H, Breslav M, Becker JM, Naider F
Peptides representing both loop and the sixth transmembrane regions of the alpha-factor receptor of Saccharomyces cerevisiae were synthesized by...
Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.
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