Urea-induced unfolding of recombinant Yarrowia lipolytica Sterol Carrier Protein-2

BURGARDT, N.; FERREIRA, R.; CÓRSICO, B.; ERMÁCORA, M.; CEOLÍN, M.

Rosario, Argentina

Congreso; XXXV Reunión anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

The sterol carrier protein-2 (SCP-2) was first identified three decades ago, but its physiological function in the lipid metabolism remains unknown. It has been proposed that SCP-2 would fulfill multiple and differentiated roles in different organisms and even in different cellular compartments. SCP-2 is present in citoplasma as in peroxisomas, interacting with membranes and enzymes, is encountered as a structural domain in several multidomain proteins and it might have functions of peroxisomal chaperone [1]. The study of the various functions proposed for SCP-2 is difficult when it coexits with other soluble lipid transporters. Yarrowia lipolytica is a very suitable system for this study, because the only known lipid binding protein in these cells is YLSCP-2, a new member of this family of proteins cloned and characterized recently in our laboratory [2]. The biochemical and biophysical properties demonstrate that rYLSCP-2 is also a good model for the structural and functional studies of the family of sterol carrier proteins. In this work, we have studied the urea induced unfolding of rYLSCP-2 monitored by intrinsic fluorescence, ANS binding, far-UV circular dicroism and small angle X-ray scattering (SAXS). The unfolding process is best adjusted to a two-state model with one transition at 1.74 M urea and a free energy change of 3.05 kcal mol-1. The results of SAXS showed that the radius of gyration increases, as the globular structure is lost. The analysis of ANS binding demonstrated that rYLSCP-2 possesses a single binding site for this ligand with an affinity of 12.8 mM. The unfolding monitored by ANS binding showed a transition at 1.44 M urea with a free energy of 1.73 kcal mol-1. This indicated that the ligand binding induces rYLSCP-2 destabilization. In this work, we present the first exhaustive characterization of a member of the SCP‑2 family. These results will be of great interest for the investigation of the molecular basis of this protein family function.