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	2001 Fellow			Cecilia Noemí Arighi, Ph.D. Found. for Adv. Education in the Sciences, Inc. Cell Biology and Metabolism Branch, NICHD Building 18T, Room 101 Bethesda, MD 20892 Tel: (301) 496-1720 Fax: (301) 402-0078 E-mail: arighic@mail.nih.gov

Country: ARGENTINA

Field: Study of the intracellular trafficking pathway of lysosomal membrane proteins using flourescent microscopic imaging techniques.

Research Interest: My current research involves the study of structural and functional aspects of the intestinal fatty acid binding protein (IFABP). This is a b-barrel protein that contains a short helix-turn-helix domain near the N-terminus, which participates in the regulation of uptake and delivery of fatty acids. NMR evidence points to an order-disorder equilibrium state at this region (Biochemistry (1997) 36, 2278). Being the 'order' conformation favored when the fatty acid is bound. To study this, we chose temperature as a physical variable to increase protein flexibility. We use aromatic residues as well as the extrinsic fluorescent probes 1,8-anilino naphthalene sulfonate and its dimer bisANS, as spectroscopic probes to monitor IFABP conformation as a function of temperature, in the absence or in the presence of its natural ligand oleic acid. In addition, given that limited proteolysis may provide information on structural changes ocurring upon binding of ligands, we also use this approach. In this way, we have detected a conformational transition, between 35 and 50°C, occurring well below the denaturation temperature of IFABP that does not involve the b-barrel structure. This transition is prevented by the binding of the fatty acid ligand. The natural ligand protects the protein from digestion supporting our previous results. An enhanced flexibility occurring at the helical domain was put forward to interpret this data, in agreement with the conformational equilibrium postulated. Currently we are conducting fluorescence and circular dichroism experiments involving the interaction of ANS and bisANS with IFABP, given that we have found a differential behavior of this probes bound to the protein in the same temperature range where the transition occurs.