Construction and analysis of a model for the transmembrane domain of the serotonin 2C G protein-coupled receptor.
Item
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Title
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Construction and analysis of a model for the transmembrane domain of the serotonin 2C G protein-coupled receptor.
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Identifier
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AAI9807903
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identifier
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9807903
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Creator
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Ballesteros, Juan Antonio.
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Contributor
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Adviser: Harel Weinstein
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Date
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1997
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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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Biophysics, Medical | Biology, Molecular | Biology, Cell
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Abstract
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A model of the transmembrane domain of the serotonin 2C G-protein coupled receptor (GPCR) complexed with its natural ligand, serotonin, has been constructed and analyzed by energetic, experimental data, and functional criteria. Interpretation of a thermodynamic framework for receptor activation in terms of energy levels for the principal states suggests a metastable activated receptor in the absence of G-protein. The modeling procedure was based on the analysis of biophysical properties and their conservation patterns observed in a multiple sequence alignment of neurotransmitter GPCRs. The boundaries and orientations of seven transmembrane {dollar}\alpha{dollar}-helices (TMH) were predicted based on the analysis of {dollar}\alpha{dollar}-helix periodicity in the conservation pattern of several biophysical properties, which can identify and discriminate between interior-facing and lipid-exposed residues. The helical periodicity of non-conserved Arg/Lys occurring at the cytoplasmic boundaries, predicted to face the mostly negatively charged phospholipid headgroups, was identified as a novel predictive tool which leads to a redefinition of a TMHs. Integration of results from the various criteria results in TMH boundaries and orientations that include most proposed G-protein coupling domains, and are in agreement with results from NMR, SCAM and Spin labeling experiments. Five organizing motifs characterize the TMH domain: The ligand binding site is connected to an "aromatic cluster" motif, characterized by a proposed "aromatic rotamer switch" in H6 which relates to receptor activation. The aromatic cluster is connected to the H2-H7-H1 cluster of polar residues, defined by involving D2.50 and N7.49 double revertant mutant constructs in the GnRH and 5HT2C receptors (Zhou 1994; Sealfon 1995). Two clusters of charged residues at the cytoplasmic boundaries, the "Arg-Cage" involving H3-H5-H6 and another cluster on H1-H2-H7, comprise the most important G-protein coupling regions. Conformational changes leading to receptor activation are described in terms of these microdomains. Highly conserved Pro-kinks are proposed as conformational hinges involved in the activation mechanism. Molecular Dynamics simulations of the 5HT2CR complex with serotonin produce an energy-refined model, which is shown to be fully consistent with available data and to have yielded specific guides for experimental verifications that validate it.
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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.
