Biophysical studies of molecular recognition in peripheral and integral membrane proteins
Item
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Title
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Biophysical studies of molecular recognition in peripheral and integral membrane proteins
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Identifier
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d_2009_2013:9672c3f612dc:11171
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identifier
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11605
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Creator
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Guan, Xudong,
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Contributor
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Ruth E. Stark
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Date
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2012
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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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Biochemistry | Biophysics | Bicelle | Docking | fatty acid-binding proteins (FABPs) | Nuclear Magnetic Resonance (NMR) | Transmembrane peptide
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Abstract
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The molecular interactions of peripheral and integral membrane proteins with ligands and model biological membranes play important roles in the regulation of human nutrition, cell signaling, and other physiological processes. In the current study, we have used solution- and solid-state nuclear magnetic resonance (NMR) and computational modeling methods to study the interactions of fatty acid-binding proteins (FABPs) with ligands and membrane mimetics, and to examine the conformation of a transmembrane peptide fragment of the Ste2p G protein-coupled receptor (GPCR) in lipids.;Computational docking with NMR-derived restraints has been used to identify critical ligand-protein electrostatic interactions in both an intermediate singly-liganded state and the double-liganded liver fatty acid-binding protein (LFABP). The model structure for a R122L/S124A LFABP mutant reveals that two charged residues are required to ensure that the first oleate ligand adopts an orientation and conformation favoring binding of the second ligand within the protein cavity. A detailed model for the ligand binding process is proposed based on the analysis of these docked structures and solution-state NMR structures determined previously.;A new isotropically tumbling bilayered micelle (bicelle) system composed of dimyristoylphosophatidylcholine (DMPC) and 1,2-di-O-hexyl-sn-glycero-3-phosphocholine (DIOHPC) has been assessed as a membrane-mimetic medium and used to probe molecular interactions with two FABPs. Rat intestinal fatty acid-binding protein (IFABP) and LFABP were titrated with the bicelles, with site-specific changes monitored by chemical shift perturbations. Contrasting transfer mechanisms for fatty acids between FABPs and membranes were differentiated based on their respective chemical shift perturbations, and site-specific information was deduced about the IFABP-membrane interaction.;Additionally, both solution- and solid-state NMR have been utilized to investigate a double transmembrane peptide (TM1TM2) from the Ste2p GPCR in phospholipid environments. A solution-state NMR interaction study of the 15N alanine-labeled peptide with DMPC-sodium docecylsulfate detergent mixtures demonstrated successful reconstitution into a helical conformation, whereas solid-state NMR experiments on TM1TM2 in DMPC multilayers permitted assignment of some signals by residue type.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Chemistry
