Characterization of fatty acid binding and transfer from Δ98Δ, a functional all-ß abridged form of IFABP.

Intestinal fatty acid binding protein (IFABP) is an intracellular lipid binding protein whose specific functions within the cell are still uncertain. An abbreviated version of IFABP encompassing residues 29-126, dubbed Δ98Δ is a stable product of limited proteolysis with clostripain of holo-IFABP. Cumulative evidence shows that Δ98Δ adopts a stable, monomeric and functional fold, with compact core and loose periphery. In agreement with previous results, this abridged variant indicates that the helical domain is-not necessary to preserve the general topology of IFABP's ß-barrel and that the helix-turn-helix motif is a fundamental element of the portal region involved in ligand binding and protein-membrane interactions. Results presented here suggest that Δ98Δ binds fatty acids with affinities lower than IFABP but higher than those shown by previous helix-less variants, shows a 'diffusional' fatty acid transfer mechanism and it interacts with artificial membranes. This work highlights the importance of the ß-barrel of IFABP for its specific functions.

Delta98Delta, a minimalist model of antiparallel beta-sheet proteins based on intestinal fatty acid binding protein.

The design of beta-barrels has always been a formidable challenge for de novo protein design. For instance, a persistent problem is posed by the intrinsic tendency to associate given by free edges. From the opposite standpoint provided by the redesign of natural motifs, we believe that the intestinal fatty acid binding protein (IFABP) framework allows room for intervention, giving rise to abridged forms from which lessons on beta-barrel architecture and stability could be learned. In this context, Delta98Delta (encompassing residues 29-126 of IFABP) emerges as a monomeric variant that folds properly, retaining functional activity, despite lacking extensive stretches involved in the closure of the beta-barrel. Spectroscopic probes (fluorescence and circular dichroism) support the existence of a form preserving the essential determinants of the parent structure, albeit endowed with enhanced flexibility. Chemical and physical perturbants reveal cooperative unfolding transitions, with evidence of significant population of intermediate species in equilibrium, structurally akin to those transiently observed in IFABP. The recognition by the natural ligand oleic acid exerts a mild stabilizing effect, being of a greater magnitude than that found for IFABP. In summary, Delta98Delta adopts a monomeric state with a compact core and a loose periphery, thus pointing to the nonintuitive notion that the integrity of the beta-barrel can indeed be compromised with no consequence on the ability to attain a native-like and functional fold.