The interactions of G-proteins with nucleotides probed by classical Raman difference spectroscopy.
Item
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Title
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The interactions of G-proteins with nucleotides probed by classical Raman difference spectroscopy.
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Identifier
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AAI9325164
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identifier
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9325164
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Creator
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Weng, Gezhi.
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Contributor
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Adviser: Robert H. Callender
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Date
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1993
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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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Physics, Molecular | Physics, Optics
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Abstract
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We explored the ligand binding pocket of Elongation Factor Tu (EF-Tu) and ras-p21, by using ultra sensitive difference Raman techniques.;The Raman difference spectra of GDP minus 8D-GDP revealed the conformation of bound GDP is c-2{dollar}\sp\prime{dollar} endo anti in both proteins.;Nucleotide analogs IDP and 6-thio-GDP are incorporated into the protein to investigate the two polar "binding handles" of the guanine base. The 2-amino and 6-keto groups are found to form strong hydrogen bonds with the proteins. The interaction of the 2-amino group with p21 is stronger than with EF-Tu, while the interaction at 6-keto position is weaker in p21 than in EF-Tu. The difference spectra of GDP minus 6-{dollar}\sp{18}{dollar}O-GDP confirmed these results.;Nucleotide, {dollar}\beta{dollar}-{dollar}\sp{18}{dollar}O{dollar}\sb4{dollar}-GDP, is used to study phosphate binding. The phosphate vibrations are found strongly affected by protein.;The difference Raman results showed that protein conformations of the active form and the inactive form are different, part of the hydrogen bonded peptide back bone has been changed. Tyrosine residue(s) and a cysteine residue of EF-Tu are involved in the conformational changes. A phosphate band, which is present in the difference spectrum of GDP minus GTP, is buried under protein changes in EF-Tu, but can be observed in p21 difference spectrum. The data give evidence that the triphosphate back bone of GTP has the same conformation in protein than in water.;Most of the results here are consistent with x-ray studies. However, Raman studies are able to give the relative strength of the hydrogen bonds, which is hard to get from x-ray results.;In the process of finding week nucleotide peaks using difference Raman spectroscopy, it was found that this technique is also very sensitive to protein hydrogen-deuterium exchanges. A few un-exchanged protons may obscure the weak nucleotide peaks of the difference Raman spectra formed between different protein-nucleotide complexes in amide I region.
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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.
