A FUNTIONAL TRUNCATED FORM OF INTESTINAL FATTY ACID BINDING PROTEIN (IFABP).

LUCRECIA MARÍA CURTO; CECILIA NOEMÍ ARIGHI; JOSÉ MARÍA DELFINO

Buenos Aires

Congreso; XIVth International Biophysics Congress (IUPAB).; 2002

IFABP (15.1 kDa) is an intracellular lipid binding protein characterized by a -clam structure built of 2 perpendicular 5-stranded -sheets and an intervening helix-turn-helix motif located in between strands A and B. This motif has been implicated in the entry of the fatty acid ligand into the binding cavity (entry portal region). Limited proteolysis of holo-IFABP (loaded with oleic acid) with clostripain (Arg-C) yields fragment 29-126 (98) as the main product revealed by SDS-PAGE. 98 (MW 10,96x by MS) is devoid of -strand A and of most of the helical domain, and also lacks the last 5 amino acids belonging to the C-terminal -strand. After separation from remnant IFABP on a MonoQ column, this peptide remains soluble. CD spectroscopy shows significant -sheet content in 98 and its intrinsic fluorescence emission spectrum (max 343.2 nm) is not unlike that of the parent protein (max 339.7 nm), lending support to the view of significant remaining structure in this fragment. These results are consistent with the conservation in 98 of all the hydrophobic amino acid residues (47,62,64,68,82,84 and 89) implicated in the nucleation event leading to the folded state. On the other hand, remarkably 98 is capable of binding a fatty acid ligand with an affinity similar (Kd~5mM for trans-parinaric acid) to that shown by a single site mutant (R106T) or a helix-less variant towards oleate (Kd~4.5mM) and approx. 100-fold higher than the wt protein. This work highlights the importance of critical structural determinants in IFABP essential for preserving its function.