“Temperature-Induced Conformational Switch in Intestinal Fatty Acid Binding Protein (IFABP) Revealing an Alternative Mode for Ligand Binding”

ARIGHI, M.C.; ROSSI, J.P.F.C.; DELFINO JM

BIOCHEMISTRY

American Chemical Society

Lugar: Vanderbilt; Año: 2003 vol. 42 p. 7539 - 7551

0006-2960

ABSTRACT: IFABP is a small â-barrel protein with a short helix-turn-helix motif near the N-terminusthat is thought to participate in the regulation of the uptake and delivery of fatty acids. In a previouswork, we detected by near UV circular dichroism a reversible conformational transition of this proteinoccurring between 35 and 50 °C in the absence of fatty acids. The addition of the natural ligand oleicacid prevents this phenomenon. In both cases, the overall structure of the â-barrel is maintained. Thisthermal transition is also detected by the fluorescent probe bis-anilino naphthalene sulfonic acid (bisANS)but not by its monomer ANS. In the present work, we studied in detail the interaction of each compoundwith IFABP as a function of temperature and in the absence or in the presence of oleic acid. A contrastingbehavior was observed for these probes: (i) IFABP is able to bind two molecules of bisANS but only onemolecule of ANS and (ii) oleic acid can fully displace ANS but only partially bisANS. Three independentlines of evidence, namely, fluorescence spectroscopy, circular dichroism, and limited proteolysis, indicatethat there is an equilibrium among different conformations of IFABP, which differ in the extent of flexibilityof the helical domain. This equilibrium can be shifted by raising temperature. bisANS is able to probe apopulation of IFABP in an altered state, which is more susceptible to cleavage by clostripain as comparedto the apo-form, whereas the conformation of IFABP bound to oleic acid is characteristically more ordered.These results highlight the idea of an enhanced flexibility exhibited by IFABP that bears importance onits transport function, supporting the role of a dynamic entry portal region for the fatty acid ligand.