NMR studies of rat liver fatty acid -binding protein (LFABP).
Item
-
Title
-
NMR studies of rat liver fatty acid -binding protein (LFABP).
-
Identifier
-
AAI9986335
-
identifier
-
9986335
-
Creator
-
He, Yan.
-
Contributor
-
Adviser: Ruth E. Stark
-
Date
-
2000
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Chemistry, Polymer
-
Abstract
-
Rat liver fatty acid-binding protein (LFABP) exhibits unique properties compared with other FABPs: it binds diverse ligands; has more than one binding site; and transfers fatty acids to membranes via aqueous diffusion rather than via direct collisional interactions. This work seeks to understand the protein functions through a study of its solution-state molecular structure and dynamics.;Sequence-specific 1H, 13C, and 15N resonance assignments were established for apo and oleate-bound holo-LFABP using multidimensional triple-resonance nuclear magnetic resonance (NMR) methods. Its secondary structural elements were determined from the consensus 1H/13C chemical shift indices. The tertiary structure of oleate-bound bolo-LFABP was calculated based on 2421 unique NMR distance constraints, yielding a family of 20 optimized structures with an average backbone RMSD of 1.05A. The LFABP conformation consists of 10 antiparallel beta-strands that form two nearly orthogonal beta-sheets of five strands each, and two short alpha-helices that connect beta-strands A and B. The NMR solution structure agrees substantially with the 2.3A X-ray crystal structure.;An oleate titration of 15N-LFABP, monitored by amide chemical shifts and linewidths in 1H-15N correlation spectra, was used to locate changes in protein structure that accompany the addition of each oleate molecule. At least two oleates (or palmitates) were bound within the LFABP cavity, based on the observation of two sets of o-CH3 and alpha-CH2 peaks in 1H-13C correlation spectra. The observation of significant linewidth differences between the spectra of holo-LFABPs with two and three equivalents of fatty acid suggested that a third oleate may also be bound to the periphery of LFABP.;H2O/D2O exchange was carried out to evaluate the solvent accessibility of apo- and holo-LFABP, revealing more extensive hydrogen bonding with ligand present. The backbone dynamics of apo- and oleate-bound bolo-LFABP were compared using amide 15N relaxation measurements, the Modelfree program was used to deduce dynamic parameters in holo-LFABP. Shorter relaxation rates, higher order parameters, and reduced solvent accessibility were found in the bolo-protein, indicating less flexibility, more order, and a more stable hydrogen-bonding network than the apo-protein.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
