CHOLESTEROL INTERACTION WITH RECOMBINANT HUMAN STEROL CARRIER PROTEIN-2

The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding methods, Lip idex 1000 binding of [H-3]cholesterol and a fluorescent dehydroergoste rol binding assay, were used to determine the affinity of SCP-2 for st erols. Binding analysis indicated SCP-2 bound [H-3]cholesterol and deh ydroergosterol with a K-d of 0.3 and 1.7 mu M, respectively, and sugge sted the presence of a single binding site. Phase fluorometry and circ ular dichroism were used to characterize the SCP-2 sterol binding site , Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime , and SCP-2 tryptophan quenching by acrylamide upon cholesterol bindin g demonstrated a shielding of the SCP-2 tryptophan from the aqueous so lvent by bound sterol. Differential polarized phase fluorometry reveal ed decreased SCP-2 tryptophan rotational correlation rime upon cholest erol binding. Circular dichroism of SCP-2 indicated that cholesterol e licited a small decrease in SCP-2 alpha helical content. The data sugg est that SCP-2 binds sterols with affinity consistent with a lipid tra ns fer protein that may act either as an aqueous carrier or at a membr ane surface to enhance sterol desorption.