INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Protein-membrane interaction and ligand transfer to membranes from intestinal fatty acid binding proteins (FABPs) employing natural ligands.
Autor/es:
EDUARDO DE GERÓNIMO*, LISANDRO J FALOMIR LOCKHART, MARÍA XIMENA GUERBI, DAVID WILTON Y BETINA CÓRSICO.
Lugar:
Montevideo, Uruguay.
Reunión:
Congreso; 6th International Conference of Biological Physics (ICBP); 2007
Resumen:
Intestinal enterocytes express large concentrations of two homologous fatty acid binding proteins (FABPs): intestinal FABP (IFABP) (15.1 kDa) and liver FABP (LFABP) (14.2 kDa). It has long been hypothesized that FABPs participate in the intracellular transport and processing of the large quantities of fatty acids (FA) absorbed by the intestine. It is currently not known why a single cell type contains two distinct types of FABP. LFABP contains at least two FA binding sites (one with higher affinity than the other) and binds a number of other endogenous hydrophobic ligands (including monoacylglycerol and acyl-CoAs) whereas IFABP has a single binding site and binds exclusively long chain FA. Based on these binding and structural differences, it has been proposed that L- and IFABP have different functions within the enterocyte. Although speculative, such hypothesis implies different mechanisms of fatty acid transfer. Using a fluorescence resonance transfer assay, it has been demonstrated that they show different rates and mechanism of anthroyloxy-fatty acid (AOFA) transfer (1). Whereas AOFA transfer from IFABP to membranes occurs by direct collisional interaction of the protein with the phospholipid bilayer, the characteristics of AOFA transfer from LFABP are slower and consistent with an aqueous diffusion-mediated process when studied under conditions of physiological ionic strength. In previous works, employing a series of structural variants (mutant and chimeric proteins) we have demonstrated the importance of the portal region of I- and LFABP in ligand binding and transfer, as well as in membrane binding (2-8), and several residues have been identified in this region as contributors to such interactions. Particularly, Leu 28 is one of the aminoacids on helix alpha-II that defines the cavity opening in LFABP and is in contact with the second FA binding site. A Trp containing mutant of LFABP at position 28 (L28W) has been analyzed for its structural and binding characteristics and proved to be remarkably effective as a probe for monitoring ligand binding (7) (Figure 1). In this work, we took advantage of the versatility of L28W to analyze natural FA transfer from LFABP to artificial membranes employing a fluorescence-based assay. To deepen our understanding on the different behavior of I- and LFABP in the transfer process, we studied FABP interaction with membranes employing photocrosslinking analysis, cytochrome c competition assay and Terbium/Dipicolinic acid complex (Tb/DPA) leakage assay.