Biosynthesis and biophysical analysis of domains of the alpha -factor receptor (Ste2p): A Saccharomyces cerevisiae G protein-coupled receptor.
Item
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Title
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Biosynthesis and biophysical analysis of domains of the alpha -factor receptor (Ste2p): A Saccharomyces cerevisiae G protein-coupled receptor.
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Identifier
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AAI3187449
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identifier
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3187449
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Creator
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Estephan, Racha.
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Contributor
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Adviser: Fred Naider
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Date
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2005
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) belongs to the G protein-coupled family of receptors (GPCRs). To analyze the structure of Ste2p a 33-residue fragment comprising the sixth transmembrane domain (M6) spanning residues 238--270 of Ste2p and a 73-mer multi-domain fragment spanning residues 267--339 containing the third extracellular loop, the seventh transmembrane domain, and 40 residues of the cytosolic tail (E3-M7-24-T40) have been biosynthesized as fusion proteins. The M6 fusion protein (M6FP), a variant M6FP(P258L), and the multidomain M7 fusion protein (M7FP) were purified to near homogeneity as judged by HPLC, and their molecular weights verified by gel electrophoresis and mass spectrometry. The 33- and the 73-residue peptides were released from their fusion proteins by CNBr and isolated via HPLC. These peptides were obtained in 14N, 15N, and 13C/15N forms in approximately 10 mg quantities.;Circular dichroism analysis of M6, M6(P258L), and E3-M7-24-T40 was performed in trifluoroethanol/water mixtures, and in the presence of detergent micelles and lipid bilayers. The M6 and M6(P258L) peptides could not be studied at concentrations higher than 100 muM in the presence of detergent micelles and did not integrate into bilayers. In contrast, the E3-M7-24-T40 peptide integrated into detergent micelles at concentrations (200--500 muM) suitable for NMR investigations and into bilayers.;HSQC experiments performed in organic-aqueous solvents and detergent micelles on the [15N]-labeled E3-M7-24-T40 peptide showed a clear dispersion of the nitrogen-amide proton correlation crosspeaks indicative of a pure, uniformly labeled molecule that assumed a partially ordered structure. 2D and 3D NMR experiments were performed in trifluoroethanol/water (1:1) and chloroform/methanol/water (4:4:1) to obtain a high-resolution structure of the 73-residue multiple domain peptide. Chemical shift indices suggested that in both aqueous-organic media helical subdomains existed in both the transmembrane and cytoslic tail of the multi-domain peptide. NMR structural modeling of the [15N]-labeled E3-M7-24-T40 peptide in both aqueous-organic media reveals a peptide with three helical parts interrupted by three proline residues. This cytosolic tail participates in down-regulation of Ste2p and the helical tendency in the cytosolic tail of the protein may play a role in protein-protein interactions leading to endocytosis and desensitization of this GPCR.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
