STRUCTURAL CHARACTERIZATION OF D98D, A MONOMERIC FUNCTIONAL ALL-B-SHEET FORM OF INTESTINAL FATTY ACID BINDING PROTEIN (IFABP)

CURTO, L.M; CARAMELO, J.J.; DELFINO, J.M.

Boston, EEUU

Congreso; 19th Symposium of The Protein Society.; 2005

IFABP is a 15 kDa intracellular lipid binding protein exhibiting a b-barrel fold that resembles a clamshell. The b-barrel, which encloses the ligand binding cavity, consists of two perpendicular five-stranded b-sheets with an intervening helix-turn-helix motif between strands A and B. D98D (fragment 29-126 of IFABP) was obtained either in its recombinant form or by limited proteolysis with clostripain. In spite of lacking extensive stretches involved in the closure of the b-barrel, D98D remains stable in solution. Spectroscopic analyses by circular dichroism, ultraviolet absorption and intrinsic fluorescence emission indicate that the fragment retains substantial b-sheet content and tertiary interactions. In particular, the environment around W82 is identical in both D98D and IFABP, a fact consistent with the conservation in the former of all the critical amino acid residues belonging to the hydrophobic core. In addition, the Stokes radius of D98D is similar to that of IFABP and 16% larger than that calculated from its molecular weight (11kDa). The monomeric status of D98D was further confirmed by chemical cross-linking experiments with disuccinimidyl suberate (DSS). Although lacking 25% of the amino acids of the parent protein, D98D unfolds through a cooperative transition with GdnHCl showing a midpoint at 0.90M. Remarkably, it also preserves binding activity for oleic acid (Kd=5µM) and for trans-parinaric acid (Kd=0.72µM). Moreover, fatty acid binding exerts a stabilizing effect on its structure. This cumulative evidence shows that D98D adopts a monomeric state with a compact core and a loose periphery, being the smallest structure of its kind preserving binding activity. Supported by CONICET, ANPCyT and UBA.