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Biochemistry

Temperature-induced conformational switch in intestinal fatty acid binding protein (IFABP) revealing an alternative mode for ligand binding.


PMID 12809510

Abstract

IFABP is a small beta-barrel protein with a short helix-turn-helix motif near the N-terminus that is thought to participate in the regulation of the uptake and delivery of fatty acids. In a previous work, we detected by near UV circular dichroism a reversible conformational transition of this protein occurring between 35 and 50 degrees C in the absence of fatty acids. The addition of the natural ligand oleic acid prevents this phenomenon. In both cases, the overall structure of the beta-barrel is maintained. This thermal 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 compound with IFABP as a function of temperature and in the absence or in the presence of oleic acid. A contrasting behavior was observed for these probes: (i) IFABP is able to bind two molecules of bisANS but only one molecule of ANS and (ii) oleic acid can fully displace ANS but only partially bisANS. Three independent lines of evidence, namely, fluorescence spectroscopy, circular dichroism, and limited proteolysis, indicate that there is an equilibrium among different conformations of IFABP, which differ in the extent of flexibility of the helical domain. This equilibrium can be shifted by raising temperature. bisANS is able to probe a population of IFABP in an altered state, which is more susceptible to cleavage by clostripain as compared to 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 on its transport function, supporting the role of a dynamic entry portal region for the fatty acid ligand.

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C0888 Clostripain from Clostridium histolyticum, ≥20 units/mg solid