Syntheses and conformational analysis of peptide pheromones and receptor fragments.
Item
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Title
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Syntheses and conformational analysis of peptide pheromones and receptor fragments.
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Identifier
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AAI9997131
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identifier
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9997131
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Creator
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Xie, Haibo.
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Contributor
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Adviser: Fred Haider
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Date
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2001
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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 | Chemistry, Organic
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Abstract
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Part I. Structure-activity relationships in the Saccharomyces cerevisiae a-factor. To investigate structure-activity relationships in the Saccharomyces cerevisiae a-factor, we have synthesized 5 analogs of a-factor, in which residues at positions 4 and 5 were replaced with Pro-containing conformation constricting sequences (compound I-V). We also prepared 3 a-factor analogs with stereoisomeric farnesyl group (compound VI-VIII with trans/cis, cis/trans and cis/cis farnesyl configurations, respectively). All analogs were purified to >99% homogeneity as evidenced by HPLC and TLC and were characterized by mass spectrometry and amino acid analysis. A growth arrest assay was employed to determine the bioactivities of the synthetic a-factor analogs. CD and NMR techniques were used to explore their secondary structures. In addition, fluorescence spectroscopy was applied to measure the partitioning coefficients of the analogs into synthetic vesicles. The results indicated that appropriate biological conformation and partitioning are both important for high activity, and they affect the activity in different ways.;Part II. Syntheses and biophysical characterization of transmembrane domains of alpha-factor receptor. The Ste2p receptor for alpha-factor, the mating pheromone of yeast Saccharomyces cerevisiae, belongs to the largest known family of cell surface receptors termed as G protein-coupled receptors (GPCRs). In this report we present the synthesis of peptides corresponding to 4 of the seven transmembrane domains (M1-M4) and 2 homologs of the sixth domain (M6). The secondary structures of all seven transmembrane peptides were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. Polyacrylamide gel electrophoresis was utilized to study the oligomeric state of the 7 fragments. Our results have implications for the folding of this membrane protein and suggest that the possible functional role of M6 is manifested through a shift in secondary structure. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.
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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.
