This project involves the study of several intracellular lipid-binding proteins from the small intestine. These proteins have distinct specificities, stoichiometries and affinities for fatty acids, steroids, retinoids and eicosanoids, but the basis for these differences is unclear. The long-term goal of this research is to define the rules that govern molecular recognition in this protein family.
The specific aims are (1) to characterize a panel of mutants designed to perturb the dynamic portal of rat and human intestinal fatty acid-binding protein (I-FABP); (2) to engineer variants of rat and human I-FABP with increased affinity for fatty acids, and to develop prototype high- affinity ligands for wild-type I-FABP; (3) to engineer a second- generation helix-less variant of I-FABP and use it as a model system for investigating the determinants of lipid-binding specificity; (4) to characterize the interactions between a physiologically relevant bile salts and ileal lipid-binding protein (I-LBP); and (5) to determine the 3D structures and backbone mobilities of human I-LBP with and without bound bile salts. The experimental approach employs triple-resonance NMR, fluorescence spectroscopy and isothermal calorimetry to characterize these proteins and their complexes with lipids. Definition of the roles for lipid-protein recognition may lead to the design of specific high affinity inhibitors with therapeutic potential in type II diabetes and hypercholesterolemia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK048046-06
Application #
6124799
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
May, Michael K
Project Start
1994-05-01
Project End
2002-11-30
Budget Start
1999-12-01
Budget End
2000-11-30
Support Year
6
Fiscal Year
2000
Total Cost
$229,342
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Horváth, Gerg?; Bencsura, Ákos; Simon, Ágnes et al. (2016) Structural determinants of ligand binding in the ternary complex of human ileal bile acid binding protein with glycocholate and glycochenodeoxycholate obtained from solution NMR. FEBS J 283:541-55
Beck, Moriah R; Dekoster, Gregory T; Cistola, David P et al. (2009) NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B. Mol Microbiol 72:344-53
Beck, Moriah R; DeKoster, Gregory T; Hambly, David M et al. (2008) Structural features responsible for the biological stability of Histoplasma's virulence factor CBP. Biochemistry 47:4427-38
Toke, Orsolya; Monsey, John D; Cistola, David P (2007) Kinetic mechanism of ligand binding in human ileal bile acid binding protein as determined by stopped-flow fluorescence analysis. Biochemistry 46:5427-36
Toke, Orsolya; Monsey, John D; DeKoster, Gregory T et al. (2006) Determinants of cooperativity and site selectivity in human ileal bile acid binding protein. Biochemistry 45:727-37
Tochtrop, Gregory P; DeKoster, Gregory T; Covey, Douglas F et al. (2004) A single hydroxyl group governs ligand site selectivity in human ileal bile acid binding protein. J Am Chem Soc 126:11024-9
Ogbay, Benhur; Dekoster, Gregory T; Cistola, David P (2004) The NMR structure of a stable and compact all-beta-sheet variant of intestinal fatty acid-binding protein. Protein Sci 13:1227-37
Tochtrop, Gregory P; Bruns, Jamie L; Tang, Changguo et al. (2003) Steroid ring hydroxylation patterns govern cooperativity in human bile acid binding protein. Biochemistry 42:11561-7
Tochtrop, Gregory P; DeKoster, Gregory T; Cistola, David P et al. (2002) A simple efficient synthesis of [23,24]-(13)C(2)-labeled bile salts as NMR probes of protein-ligand interactions. Bioorg Med Chem Lett 12:433-5
Tochtrop, Gregory P; Richter, Klaus; Tang, Changguo et al. (2002) Energetics by NMR: site-specific binding in a positively cooperative system. Proc Natl Acad Sci U S A 99:1847-52

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