CONICET | Buscador de Institutos y Recursos Humanos

Fatty acid transfer from intestinal fatty acid-binding protein(IFABP) to phospholipid membranes occurs during protein-membranecollisions. Electrostatic interactions involving the -helicalportal region of the protein have been shown to be of great importance.In the present study, the role of specific lysine residues in the-helical region of IFABP was directly examined. A series of pointmutants in rat IFABP was engineered in which the lysine positivecharges in this domain were eliminated or reversed. Using a fluorescenceresonance energy transfer assay, we analyzed the rates andmechanism of fatty acid transfer from wild type and mutant proteinsto acceptor membranes. Most of the-helical domain mutantsshowed slower absolute fatty acid transfer rates to zwitterionicmembranes, with substitution of one of the lysines of the 2 helix,Lys27, resulting in a particularly dramatic decrease in the fatty acidtransfer rate. Sensitivity to negatively charged phospholipid membraneswas also reduced, with charge reversal mutants in the 2helix the most affected. The results support the hypothesis that theportal region undergoes a conformational change during proteinmembraneinteraction, which leads to release of the bound fattyacid to the membrane and that the 2 segment is of particularimportance in the establishment of charge-charge interactionsbetween IFABP and membranes. Cross-linking experiments with aphospholipid-photoactivable reagent underscored the importanceof charge-charge interactions, showing that the physical interactionbetween wild-type intestinal fatty acid-binding protein and phospholipidmembranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by thepresence of ligand, suggesting different collisional complex structures