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

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

Montevideo, Uruguay,

Congreso; 6th International Conference of Biological Physics ICBP 2007, 5th Southern Cone Biophysics Congress, 34th Annual Meeting of Argentinean Biophysical Society (SAB).; 2007

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 stable and functional variant (98) by limited proteolysis of IFABP with clostripain (Curto et al., 2005). To probe the tertiary structure of 98, we evaluated the proteolytic pattern of this variant in the presence (holo form) and in the absence (apo form) of the ligand. The results revealed that the holo form is much more resistant to proteolysis than the apo form, yielding a major product corresponding to a smaller fragment, herein referred to as 78 (corresponding to residues 29-106 of the full length protein). By comparison with 98, this fragment lacks all the alpha helical domain plus beta strand A, and a stretch comprising the last 25 residues. The aim of this work is to biophysically characterize this new abridged variant 78 to check 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. Identification of 78 was achieved after its molecular weight (8.8 kDa) was determined by ES mass spectrometry. Structural analyses - by CD, UV absorption and fluorescence spectroscopies - indicate 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 analyses - by cross-linking experiments carried out with DSS and dynamic light scattering - confirm that 78 gives rise to a dimer. Binding experiments with oleic acid reveal that, although showing lower affinity (0.17 M) than the wild-type protein (0.06 M), 78 still preserves ligand binding capacity. Taken together, this body of evidence points to a substantial structural plasticity inherent to this beta barrel motif that does not compromise the binding function.