Determinants of stability and stoichiometry in intestinal fatty acid binding protein.

摘要

Intestinal fatty acid binding protein (I-FABP) is a member of intracellular lipid-binding protein family that bind fatty acids, retinoids, and sterols. Its structure adopts a β-clam topology that contains a sequestered interior ligand-binding cavity. It binds long chain fatty acids at high affinity, and is thought to function in the facilitation of intracellular lipid transfer.; This thesis project had two main goals: (i) designing a stable and compact helix-less variant of I-FABP that could be used as a scaffold for the design of artificial enzymes or drug carriers, and (ii) investigating the determinants of ligand binding stoichiometry in I-FABP. Two crucial factors that apparently define the stoichiometry were examined: (a) the length of the sequence that links the first two β-strands in the structure of I-FABP, and (b) the ion-pair interaction between the side-chains of D34 and R126.; Using recombinant DNA techniques, we engineered a stable second-generation helix-less variant (Δ27-GG). Its three-dimensional structure and ligand binding properties were characterized using NMR and fluorescence spectroscopy. Ligand binding studies showed that Δ27-GG has peculiar fatty acid binding properties in that it binds more than three fatty acid molecules, in stark contrast to the wild-type protein which was previously though to bind only one. Careful examination of the wild-type protein revealed a second, previously unidentified binding site.; To investigate the determinants of stoichiometry in I-FABP, a series of helix-less variants with and without a D34A mutation, as well as D34A and R126A single-site mutants of I-FABP were engineered. Characterization of the ligand binding properties of these proteins revealed that perturbation of the D34-R126 ion-pair interaction increases the number of binding sites by one. Moreover, deletion of the helical domain and shortening of the loop that connects the first two β-strands in I-FABP further increases the binding stoichiometry by one.