Effect of R122L/S124A and K31L mutations on molecular structure and function in rat liver fatty acid -binding protein.
Item
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Title
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Effect of R122L/S124A and K31L mutations on molecular structure and function in rat liver fatty acid -binding protein.
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Identifier
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AAI3187447
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identifier
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3187447
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Creator
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Estephan, Rima.
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Contributor
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Adviser: Ruth E. Stark
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Date
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2005
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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, Biochemistry | Biophysics, General
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Abstract
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The unique binding and transfer mechanism of LFABP among proteins of the iLBP family have motivated the construction of two mutants: K31L modifies the charged residue in the first helix that is thought to interact with the first FA and to partially account for the transfer mechanism; R122L/S124A alters the binding cavity where Arg122 and Ser124 interact with the second FA. Titration and binding studies suggested that unlike K31L-LFABP, the R122L/S124A-LFABP mutant may bind just one fatty acid with a much lower affinity. Further NMR analysis of intermolecular NOE data between the ligand (oleate) and R122L/S124A-LFABP revealed that there is one oleate binding: the carboxylate end interacts with K96 and I98 near the opening of the beta-barrel, and the hydrocarbon tail interacts with S39, I41, T51, and I109 in the cavity.;NMR-based chemical shift perturbation analysis revealed localized structural perturbations for the K31L mutant but more pervasive conformational changes for R122L/S124A. Nevertheless, neither of these mutations altered the transfer rate of FA to membranes. The solution secondary structures, based on chemical shift indices for 1Halpha, 13C alpha, and 13Cbeta, sites, indicated 10 beta-strands and 2 alpha-helices as found for wild type-LFABP.;Interproton distance restraints derived from 3D and 4D nuclear Overhauser effect NMR experiments were used to calculate the best-fit tertiary structures of apo- and holo-R122L/S124A-LFABP. The apo-R122L/S124A-LFABP three-dimensional structures was well-defined with average pairwise RMSDs, for the 10 lowest energy structures, of 0.47 and 0.91 A for backbone and all heavy atoms, respectively. However, the two alpha-helices are closer to the beta-barrel in the mutant than in the wild-type protein forming a more closed portal that could explain the lower on-rate of the ligand.;The holo-R122L/S124A-LFABP preliminary three-dimensional structure was very similar (b.b. RMSD = 0.64 A) to that of the apo-R122L/S124A-LFABP. Compared to the holo wild-type protein, one half of the beta-clam (betaG, betaH, betaI, and betaJ) is superimposable with that of the holo wild-type structure, but the other half of the beta-clam (betaA, betaB, betaC, betaD, betaE and betaF) is displaced. In addition, the overall spatial relationship of the helix motif relative to the beta-clam is similar to that in the holo wild-type structure. The three-dimensional structure of R122L/S124A-LFABP complexed with oleate is being further refined using both intermolecular NOE measurements and protein restraints.
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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.
