Tools for Functional Analysis of Soluble Lipid Binding Proteis

FALOMIR LOCKHART, LISANDRO J.; FRANCHINI, GISELA R; STORCH, JUDITH; CÓRSICO, BETINA

Buenos Aires, Argentina

Workshop; Advances in technologies for high resolution in vivo microscopy: From single molecule detection to functional studies; 2006

CMA/Fac. Cs. Exactas (UBA)/EMBO

Fatty Acid Binding Proteins (FABP) are small soluble cytosolic proteins of about 15 kDa. They belong to the family of soluble Lipid Binding Proteins, which shows a very similar tertiary structure with homologies as low as 20%. The crystal structure shows 10 antiparallel beta-sheets forming a barrel and a helix-turn-helix motif serving as a cap of the barrel. Two different isofomrs of FABP are been expressed in the intestinal epithelium, Liver FABP (LFABP or FABP4) and Intestinal FABP (IFABP or FABP2). It has been long known that LFABP and IFABP show different apparent mechanism to transfer ligands to model membranes. While IFABP seems to transfer fatty acids through a collisional mechanism, LFABP apparently employs a diffusional mechanism where the interaction with the membranes is not the rate-limiting step. Employing different fluorescence techniques, we have demonstrated the importance of the alpha-helical domain in the determination of the transfer mechanism and, particularly, the relevance of the positively charged residues in this region; as well as the ability of these proteins to interact with model unillamelar phospholipids membranes. We used a FRET assay to study the transfer mechanism and rates of antroyloxy derivatives of fatty acids to model membranes harbouring the quencher NBD-PC. On the other hand, a binding competition assay with Cytochrome c and a leakage assay of Tb/DPA complex were used to study the physical interaction of FABPs with membranes. The results obtained with the wild type proteins and several structural variants (mutants and chimeras) shows new differences to the ones already observed between LFABP and IFABP, supporting the idea that the different isofomrs should have specific and distinctive roles in the fatty acid traffic and metabolism inside the enterocytes.