INTESTINAL FATTY ACID BINDING PROTEINS

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Intestinal enterocytes express high levels of two homologous fatty acid-binding proteins (FABP), liver FABP (LFABP) and intestinal FABP (IFABP). It is hypothesized that FABPs are important in intracellular transport of FA. Both I- and LFABP bind long chain fatty acids (FA) with high affinity, and it has recently been shown that they have similar tertiary structures. Nevertheless, it has been suggested that they are functionally distinct because they have different substrate specificities and stoichometry and different tissue distributions. Furthermore, recent work from our laboratory has shown that these proteins employ markedly different mechanisms to transfer FA to acceptor membranes. Whereas FA transfer from LFABP occurs via aqueous diffusion, FA transfer from IFABP occurs during direct collisional interactions of the protein with acceptor membranes. We have recently demonstrated the critical importance of the helix-turn helix domain in the interaction of IFABP with membranes, and in its FA transfer mechanism. In the present proposal we will use biochemical, molecular biological and biophysical techniques to provide a molecular level analysis of the specific domains and residues that are involved in these IFABP-membrane interactions, as well as a detailed understanding of the physicochemical nature of the IFABP-membrane association itself. Further, the structural basis for the absence of collisional transfer of FA from LFABP will also be elucidated. The specific aims of this proposal are: 1) To determine the importance of the alphaI helix and the alphaII helix in collisional transfer of fatty acids to membranes, by constructing chimeric I/LFABP; and 2) To elucidate the roles of specific charged and hydrophobic residues in collisional transfer of FA to membranes, employing site-directed mutagenesis techniques. This approach to the fatty acid transport function of FABPs should provide molecular level detail of the specific structural elements which lead to the unique transport properties of IFABP versus LFABP. Specifically, for all point mutant and chimeric proteins engineered, we will analyze the rate and mechanism of fatty acid transfer to model membranes using a fluorescence resonance energy transfer assay, and the interaction of the proteins with membranes using several complementary biochemical and biophysical techniques. Since both LFABP and IFABP are highly expressed in the small intestinal enterocyte, an understanding of their mechanisms of actions will enable the effective nutritional and/or pharmacological manipulation of dietary lipid assimilation.