Delta-98-Delta, a functional all-beta-sheet abridged form of intestinal fatty acid binding protein

CURTO LM; CARAMELO JJ; DELFINO JM

BIOCHEMISTRY

AMER CHEMICAL SOC

Año: 2005 vol. 44 p. 13847 - 13857

0006-2960

Intestinal fatty acid binding protein (IFABP) is a 15 kDa intracellular lipid-binding protein exhibiting a beta-barrel fold that resembles a clamshell. The beta-barrel, which encloses the ligand binding cavity, consists of two perpendicular five-stranded beta-sheets with an intervening helix-turn-helix motif between strands A and B. ∆98∆ (fragment 29-126 of IFABP) was obtained either in its recombinant form or by limited proteolysis with clostripain. Despite lacking extensive stretches involved in the closure of the -barrel, ∆98∆ remains soluble and stable in solution. Spectroscopic analyses by circular dichroism, ultraviolet absorption, and intrinsic fluorescence indicate that the fragment retains substantial beta-sheet content and tertiary interactions. In particular, the environment around W82 is identical in both ∆98∆ 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 ∆98∆ is similar to that of IFABP and 16% larger than that calculated from its molecular weight (11 kDa). The monomeric status of ∆98∆ was further confirmed by chemical cross-linking experiments. Although lacking 25% of the amino acids of the parent protein, in the presence of GdnHCl, ∆98∆ unfolds through a cooperative transition showing a midpoint at 0.90 M. Remarkably, it also preserves binding activity for fatty acids (Kd ) 5.1 ìM for oleic acid and Kd ) 0.72 ìM for trans-parinaric acid), a fact that exerts a stabilizing effect on its structure. These cumulative evidences show that ∆98∆ adopts a monomeric state with a compact core and a loose periphery, being so far the smallest structure of its kind preserving binding function.