STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE NEW ABRIDGED VARIANT (D78D) OF THE INTESTINAL FATTY ACID BINDING PROTEIN (IFABP)

GISELA R. FRANCHINI; LUCRECIA M. CURTO; JULIO J. CARAMELO; JOSÉ M. DELFINO

Rosario, Santa Fe

Congreso; XXXV Reunion Anual de la Sociedad Argentina de Biofisica (SAB); 2006

IFABP is a 15 kDa intracellular lipid binding protein that represents an excellent model of study for beta barrel proteins. It exhibits a β-clam structure, which encloses the ligand binding cavity, built of 2 perpendicular 5-stranded β-sheets and an intervening helix-turn-helix motif located in between strands A and B. Recently, we produced a functional variant (98) by limited proteolysis of IFABP with clostripain (1). Despite the lack of β-strand A, most of the helical domain and the last 5 C-terminal amino acids, 98 remains stable and soluble. To check for conformational changes in the tertiary structure of 98, we evaluated the proteolytic pattern of this variant in the presence and in the absence of the ligand fatty acid. The results revealed that the holo form (in the presence of ligand) is much more resistant to proteolysis than the apo form (in the absence of ligand), yielding a major product corresponding to a smaller fragment denominated 78 (corresponding to residues 29-106 of the full length protein). By comparison with 98, this fragment lacks the whole alpha helical domain plus beta strand A and the last 25 C-terminal residues. The aim of this work is to characterize this new abbreviated variant (78) to see whether it remains functional and properly folded by comparison with IFABP. To this end, 78 was cloned in the prokaryotic vector pET-11d, and a straightforward purification protocol was devised. Specifically, we recovered pure protein from inclusion bodies solubilized in 2 M urea and passage through a Superdex 75 column. Identification of 78 was achieved after its molecular weight was determined by mass spectrometry. It corresponds to a 8.8 kDa fragment, still conserving binding function. Structural analysis - by CD, UV absorption and fluorescence spectroscopies - indicates that 78 retains substantial beta-sheet content and tertiary interactions. By comparison with 98 and IFABP, the Superdex-75 elution profile of apo-78 exhibits a behavior corresponding to a protein of a significantly larger Stokes radius than that expected from its molecular weight. Further analysis by cross-linking experiments run with DSS reveals a clear propensity to form a dimer. Taken together, this body of evidence points to the substantial structural plasticity inherent to this beta barrel motif that does not compromise binding function.