Study of cyclic alpha-factor analogs of the yeast Saccharomyces cerevisiae.
Item
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Title
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Study of cyclic alpha-factor analogs of the yeast Saccharomyces cerevisiae.
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Identifier
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AAI9605687
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identifier
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9605687
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Creator
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Yang, Wei.
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Contributor
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Adviser: Fred Naider
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Date
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1995
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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, Polymer | Chemistry, Biochemistry
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Abstract
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Previous biophysical studies on the {dollar}\alpha{dollar}-factor mating pheromone (WHWLQLKPGQPMY) of Saccharomyces cerevisiae suggested that this peptide adopted a type II {dollar}\beta{dollar}-turn in DMSO, water or in the presence of lipid vesicles (Jelicks et al., 1988; Jelicks et al., 1989). The results of studies on linear and cyclic {dollar}\alpha{dollar}-factor analogs by Gounarides et al. (1993; 1994) indicated a type II {dollar}\beta{dollar}-turn spanning residues 7 and 10 in active analogs such as {dollar}\lbrack D{dollar}-Ala{dollar}\sp9\rbrack\alpha{dollar}-factor or cyclo{dollar}\sp{lcub}7,10{rcub}\rbrack\rm Cys\sp{lcub}7,10{rcub},{lcub}\it D{rcub}{dollar}-Ala{dollar}\rm \sp9,Nle\sp{12}\rbrack\alpha{dollar}-factor but not in the less active analogs such as {dollar}\lbrack L{dollar}-Ala{dollar}\sp9\rbrack\alpha{dollar}-factor or cyclo{dollar}\sp{lcub}7,10{rcub}\lbrack\rm Cys\sp{lcub}7,10{rcub},{lcub}\it L{rcub}{dollar}-Ala{dollar}\rm \sp9,Nle\sp{12}\rbrack\alpha{dollar}-factor. To further understand the conformational properties of the central region in {dollar}\alpha{dollar}-factor, we synthesized eight cyclic analogs and their corresponding linear homologs. The cyclic analogs contained a lactam ring formed from the side chains of residues 7 and 10, and the size of the ring was systematically varied from 14 to 18 atoms. All peptides were synthesized using the solid phase method and purified to high homogeneity (over 99% pure as judged by acetonitrile/water/trifluoro-acetic acid gradients on reversed-phase HPLC). The chemical structures of the analogs were characterized by FABMS, amino acid analysis and NMR spectroscopy. The biological activities of the cyclic analogs varied from 10% to less than 0.1% of that of {dollar}\lbrack\rm Nle\sp{12}\rbrack\alpha{dollar}-factor against strain RC629 (sstl) of S. cerevisiae. Except for the Orn{dollar}\sp7{dollar}-containing cyclic peptides (C32 and C31), analogs with Glu in position 10 were more active than the homologs with Asp at this position. The bioactivity results suggested that the {dollar}\gamma{dollar}-carbonyl group of residue 10 is a critical determinant of the biological activity.;The conformations of the cyclic {dollar}\alpha{dollar}-factor analogs were examined in a water/methanol (1:1, v/v) solvent mixture. For comparison CD spectra of the linear analogs were acquired in the same solvent. All linear analogs showed similar unstructured CD spectra. In contrast, the CD spectra of the cyclic analogs appeared quite different from each other. Except for C12 and C(4)2, a positive band below 200 nm and a minimum between {dollar}\sim{dollar}200 and 215 nm were observed in the CD spectra of the cyclic analogs. Such spectral features were not observed in the CD spectra of the linear homologs. CD studies on model cyclic tetrapeptides (Tetra 42 and Tetra 32) and examination of solutions containing both the linear tridecapeptide (L32) and Tetra 32 suggested that the constrained region from residues 7 to 10 in the cyclic analog (C32) retained the same conformation as the model cyclic tetrapeptide,;Two cyclic {dollar}\alpha{dollar}-factor analogs, C42 and C32, were analyzed by NMR techniques. NOE, NH d{dollar}\delta{dollar}/dT and {dollar}\sp3\rm J\sb{lcub}\alpha NH{rcub}{dollar} coupling constants suggested that in DMSO solution C32 adopts a type II {dollar}\beta{dollar}-turn spanning residues 7-10 with some distortion, and C42 exists as mixture of a type II {dollar}\beta{dollar}-turn centered around the Pro{dollar}\sp8{dollar}-Gly{dollar}\sp9{dollar} sequence and a {dollar}\gamma{dollar}-turn around Pro{dollar}\sp8{dollar}. The Corey-Pauling-Koltun (CPK) model building based on the NOE constraints and amide proton temperature dependence revealed the importance of spatial orientation of the Glu{dollar}\sp{10}\gamma{dollar}-carbonyl from a conformational point of view. This observation is in agreement with the results of our bioactivity analysis.
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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.
