FURTHER STRUCTURAL CHARACTERIZATION OF D98D A FUNCTIONAL ABRIDGED FORM OF THE INTESTINAL FATTY ACID BINDING PROTEIN (IFABP).

LUCRECIA MA. CURTO; JULIO J. CARAMELO; JOSÉ MA. DELFINO

Rosario, Santa Fé

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica (SAB); 2006

98 (11 kDa) is a functional abridged variant of the intestinal fatty acid binding protein (IFABP). The parent protein is a 15 kDa intracellular lipid binding protein exhibiting a -barrel fold that resembles a clamshell. The -barrel, which encloses the ligand binding cavity, consists of two perpendicular five-stranded -sheets with an intervening helix-turn-helix motif between strands A and B. 98 is devoid of -strand A, most of the helical domain and the last 5 C-terminal amino acids (fragment 29-126 of IFABP). Despite the lack of extensive stretches involved in the closure of the -barrel, cumulative evidence including circular dichroism, fluorescence spectroscopy, gel filtration chromatography and chemical cross-linking experiments, showed that 98 adopts a monomeric state with a compact core and a loose periphery (1). The accessibility to the only remaining W in the abridged form (W82, which is buried in the hydrophobic core of IFABP) was studied by the quenching of its fluorescence. As temperature rises, the Stern-Volmer constant (Ksv) for acrylamide of apo- and holo-98 increases substantially, by contrast with IFABP which shows only a minor effect. In the same fashion, the reduction in the intrinsic fluorescence of 98 caused by KI is greater than that observed for IFABP. Taken this evidence together in the abridged variant the accessibility of W82 appears to be less sterically restricted. Unlike most native proteins, IFABP naturally binds anilino naphthalene sulfonic acid (ANS). By contrast to IFABP, that binds only one molecule of this fluorophore, 98 binds two molecules. However, the environment of the additional putative binding site seems to be more hydrophobic than that shared with IFABP. Conformational stability of the new variant was tested by circular dichroism and fluorescence spectroscopy. 98 unfolds along a cooperative transition either with GdnHCl (showing a midpoint at 1.14M) or heat (Tm ~71 °C). Moreover, fatty acid binding exerts a slight stabilizing effect on its structure. Exclusion chromatography (S-75) of 98 run in the presence of a saturating concentration of oleic acid indicates a shift toward a discrete high-molecular size species. Nevertheless, cross-linking with DSS does not reveal pre-formed aggregates. Thus, under these conditions, the fragment probably adopts a structure resembling a lipoproteic particle.