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Author Francis, Fouad
Title NMR structural studies of fatty acid-binding proteins
book jacket
Descript 162 p
Note Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5928
Adviser: Ruth Stark
Thesis (Ph.D.)--City University of New York, 2007
Solution-state NMR has been used to assess the binding of L-FABP to various ligands that differ in size, chain length, and hydrophobicity. 15N HSQC NMR spectra of holo-L-FABPs containing two ligand equivalents display large chemical shift perturbations for long-chain fatty acids such as oleate, palmitate, eicosapentaenoate, as well as lysophosphatidylcholine, when compared with the unliganded (apo) protein. Similar perturbation patterns for oleate, palmitate, and lysophosphatidylcholine, suggest that binding of these ligands occurs in analogous regions within the binding cavity of L-FABP. However, a difference in the chemical shift perturbation for eicosapentaenoate residues suggests a different binding orientation. By contrast, the short-chain fatty acid caprylate causes a more modest perturbation primarily in the portal region, supporting a shallow interaction with respect to the cavity and yielding K d values of 0.3-2.2 mM measured at various protein sites. Addition of both heme and protoporphyrin, previously reported as strongly binding ligands for L-FABP, causes protein denaturation. However, heme denaturation can be reversed by the addition of oleate. Absence of any changes in the HSQC spectra of apo-LFABP with water-insoluble cholesterol and observance of considerable changes with water-soluble deoxycholate (bile salt) suggests an electrostatic requirement for sterol binding
Introducing residual dipolar couplings (RDC) as angular constraints achieves refinement of the solution-state tertiary structure of apo-L-FABP, improving values of backbone RMSD from 1.04 to 0.72 for apo-L-FABP. In addition, a better apo-L-FABP structure refined with RDCs is indicated by a decrease in the percentage of disallowed regions (1.2 to 0.2%) in the Ramachandran plot. In particular, unambiguous orientations for residues forming the portal "lid" region of the protein were determined, suggesting a more open conformation for the portal region of apo-L-FABP as compared with the holo protein
Isotropically tumbling bicelles (q = 0.5) were used as mimetic media for lipid membranes in the study of protein-membrane interactions of both L-FABP and I-FABP with high-resolution NMR. HSQC titration spectra for both proteins were compared with their apo counterparts. The largest chemical shift perturbations were located in the portal region (helix II) of both L-FABP and I-FABP, in addition to the C-D and E-F turns for L-FABP, and C-D and E-F beta strands for I-FABP. Preparation of a stable protein-bicelle complex permitted sequence-specific resonance assignments to be made for a model membrane-associated I-FABP system
School code: 0046
Host Item Dissertation Abstracts International 68-09B
Subject Chemistry, Biochemistry
Alt Author City University of New York. Biochemistry
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