Role of the Sterol Carrier Protein-2 N-Terminal Membrane Binding Domain in Sterol Transfer

Previous studies showed that the N-terminal 32 amino acids of sterol carrier protein-2 (1-32SCP2) comprise an amphipathic α-helix essential for SCP2 binding to membranes [Huang et al. (1999) Biochemistry 38, 13231]. However, it is unclear whether membrane interaction of the 1-32SCP2 portion of SCP2 is in itself sufficient to mediate intermembrane sterol transfer, possibly by altering membrane structure. In this study a fluorescent sterol exchange assay was used to resolve these issues and demonstrated that the SCP2 N-terminal peptide 1-32SCP2 did not by itself enhance intermembrane sterol transfer but potentiated the ability of the SCP2 protein to stimulate sterol transfer. Compared with SCP2 acting alone, 1-32SCP2 potentiated the sterol transfer activity of SCP2 by increasing the initial rate of sterol transfer by 2.9-fold and by decreasing the half-time of sterol transfer by 10-fold (from 11.6 to 1.2 min) without altering the size of the transferable fractions. The ability of a series of SCP2 mutant N-terminal peptides to potentiate SCP2-mediated sterol transfer was directly correlated with membrane affinity of the respective peptide. N-Terminal peptide 1-32SCP2 did not potentiate intermembrane sterol transfer by binding sterol (dehydroergosterol), altering membrane fluidity (diphenylhexatriene) or membrane permeability (leakage assay). Instead, fluorescence lifetime measurements suggested that SCP2 and 1-32SCP2 bound to membranes and thereby elicited a shift in membrane sterol microenvironment to become more polar. In summary, these data for the first time showed that while the N-terminal membrane binding domain of SCP2 was itself inactive in mediating intermembrane sterol transfer, it nevertheless potentiated the ability of SCP2 to enhance sterol transfer.