Circular dichroism and fluorescence spectroscopy studies of the protein synthesis initiation factors withmRNA.
Item
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Title
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Circular dichroism and fluorescence spectroscopy studies of the protein synthesis initiation factors withmRNA.
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Identifier
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AAI9630517
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identifier
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9630517
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Creator
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Wang, Yahong.
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Contributor
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Adviser: Dixie J. Goss
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Date
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1996
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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, Analytical | Chemistry, Biochemistry
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Abstract
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The structural features of wheat germ protein synthesis initiation factor eIF-(iso)4F, which has a cap binding protein as one of its two subunits, are unknown. In this study, circular dichroism (CD) spectra and secondary structure prediction were obtained for eIF-(iso)4F and its two subunits, p28 and p86. The {dollar}\alpha{dollar}-helix content changed from 42% at pH 6.3 to 15% at pH 7.6, the optimum pH for cap binding. The {dollar}\beta{dollar}-sheet content increased from 14% at pH 6.3 to 38% at pH 7.6. The CD spectra of the two subunits, p28 and p86 were also measured and analyzed. The separated subunits both had a higher {dollar}\alpha{dollar}-helix content at pH 7.6 than the native protein, giving values of 60% and 34% {dollar}\alpha{dollar}-helix for p28 and p86, respectively. Binding of the dinucleotide cap analog to p28 reduced the {dollar}\alpha{dollar}-helix content to approximately 8% with an increase in the {dollar}\beta{dollar} sheet content from 10% to 37%. In order to characterize the structural changes of wheat germ protein synthesis initiation factor elF-(iso)4F and p28 binding with mRNA cap analogs, CD spectroscopy was also performed on this system as a function of pH. The results reveal that the conformational changes in eIF-(iso)4F upon binding with mRNA are dependent on cap or oligonucleotide structure. A conformation consisting of approximately the same {dollar}\alpha{dollar}-helix and {dollar}\beta{dollar}-sheet content can be induced by ligands even at non-optimal pHs. This large conformational transition suggests eIF-(iso)4F binds nucleic acids by interaction of a {dollar}\beta{dollar}-sheet motif and that these conformational transitions may have a regulatory role. The fluorescence stopped-flow studies of eIF-(iso)4F and p28 with cap show a concentration-independent conformational change. The rate of this conformational change was approximately ten fold faster for the isolated p28 compared to the native eIF-(iso)4F.;Recently the purified subunit p26, 26 kDa of wheat germ protein synthesis initiation factor eIF-4F has been obtained from E. coli expression of the cloned DNA. CD spectroscopy revealed 15% {dollar}\alpha{dollar}-helix and 36% {dollar}\beta{dollar}-sheet in p26, and 38% {dollar}\alpha{dollar}-helix and 17% {dollar}\beta{dollar}-sheet in eIF-4F. The binding of the 5{dollar}\sp\prime{dollar}-terminal cap analog m{dollar}\sp7{dollar}GTP to p26 as a function of pH and temperature was described. The optimum binding pH was 8.0, {dollar}\Delta{dollar}H and {dollar}\Delta{dollar}S were 10.23 kcal/mol and 57.33 cal/mol{dollar}\sp\circ{dollar}C, respectively. The environment of tryptophan residues in p26 and eIF-4F were monitored by using various quenchers. The results indicated the presence of negatively charged residues near the tryptophans which were surrounded by a hydrophobic environment in p26 and eIF-4F. The stopped-flow kinetics demonstrated the interaction of eIF-4F with m{dollar}\sp7{dollar}GpppG cap analog followed a two-step mechanism. The rate constant and the activation energy for the second step were 121.7 s{dollar}\sp{lcub}-1{rcub}{dollar} and 4.55 kcal/mol, respectively.
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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.
