The long term objective is to establish the basis for molecular recognition in the family of intracellular lipid-binding proteins (iLBPs). The small intestine expresses four iLBP variants with similar tertiary structures but distinct ligand specificities. Intestinal fatty acid-binding protein (I-FABP) binds long-chain fatty acids while ileal lipid-binding protein (I-LBP) preferentially binds bile salts. We hypothesize wild type I-FABP possesses most of the structural and dynamical features necessary for bile salt binding, but lacks a few specific side chains required for favorable pairwise interactions.
Specific Aim I is to engineer, express and purify mutant variants of I-FABP with increased affinity for bile salts. Bile salt binding will be assayed using fluorescence spectroscopy, isothermal titration calorimetry and site-specific NMR.
Aim II is to characterize the structure of a selected bile salt/protein complex in solution using triple-resonance 3-D and 4-D NMR methods. This structural and dynamical analysis will reveal the identity of side-chains involved in bile salt/protein contacts and indicate whether chances in the conformation or flexibility of I-FABP are necessary for bile salt binding. Blockage of bile salt reabsorption by the small intestine lowers plasma cholesterol, and thus, the iLBPs represent potential targets for the design of pharmaceutical agents to treat hypercholesterolemia. The proposed work will ultimately facilitate the design of such agents.
| 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 |
