D98D, A FUNTIONAL ALL-B-SHEET ABRIDGED FORM OF INTESTINAL FATTY ACID BINDING PROTEIN (IFABP).

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

Angra do Reis

Congreso; 1st Latin American Protein Society Meeting.; 2004

IFABP is a 15kDa intracellular lipid binding protein exhibiting a -clam structure built of 2 perpendicular 5-stranded -sheets and an intervening helix-turn-helix motif located in between strands A and B. 98 (fragment 29-126) is obtained by limited proteolysis of IFABP with clostripain. In spite of the lack of -strand A, most of the helical domain and the last 5 C-terminal amino acids, 98 remains stable and soluble. Significantly, distance maps reveal that stretch 1-28 in IFABP encompasses a region with the least number of neighboring amino acids in the whole protein, whereas contact analysis expose an imperfect hydrogen bonding network between strand A and adjacent strands B and J. Thus, 98 is devoid of both partners responsible for closing the -barrel in IFABP. Structural analysis by CD, UV absorption and fluorescence spectroscopies indicates that this fragment retains substantial -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, i.e. those implicated in the nucleation event leading to the folded state. Apo 98 (the species free from fatty acids) shows a Stokes radius 30% larger than that expected from its molecular weight (11kDa), and a somewhat less cooperative unfolding behavior (with a midpoint at 3.1 M urea) as compared to IFABP. The presence of the fatty acid ligand exerts a stabilizing effect on the structure. Remarkably, this fragment does not exhibit larger propensity to oligomerize than the parent protein, as shown by covalent cross-linking experiments. This cumulative evidence led us to infer that 98 would adopt a monomeric state endowed with a well-folded core but expanded in its periphery. From a functional standpoint, 98 retains the ability to interact with several amphipathic ligands, representing the smallest structure of its kind preserving structure and binding activity.