Binding of steroidogenic acute regulatory protein to synthetic membranes suggests an active molten globule

Steroidogenic acute regulatory protein (StAR) mediates cholesterol transpor t from the outer to the inner mitochondrial membrane during steroid biosynt hesis, The mechanism of StAR's action is not established, To address mechan istic issues, we assessed the binding of StAR to artificial membranes by fl uorescence resonance energy transfer using endogenous StAR tryptophan resid ues as the donor and dansyl-phosphatidylethanolamine in the bilayer as the acceptor. Mixing StAR with dansyl-labeled vesicles composed of phosphatidyl choline increased the fluorescence intensity of dansyl emission excited at 280 nm by 10-40%, This interaction was dependent on pH, with a maximum at p H 3.0-3.5 and essentially no change above pH 5, Binding experiments at diff erent temperatures and various combinations of phosphatidylcholine, phospha tidylglycerol, cardiolipin, and cholesterol showed that binding involves an electrostatic step and one or more other steps. Although binding prefers a thermodynamically ordered bilayer, the rate-limiting step occurs either wh en the bilayer is in a fluid state or when there is cholesterol-induced mem brane heterogeneity, Experiments with fluorescence and light scattering ind icate that StAR binding promotes ordering and aggregation of anionic membra nes. The inactive StAR mutant R182L had lower affinity for the membrane, an d the partially active mutant L275P had intermediate affinity. Far-UV CD sp ectroscopy of StAR in PC membranes show more p-structure than in aqueous bu ffers, and the presence of cardiolipin or cholesterol in the membrane foste rs a molten globule state. Our data suggest that StAR binds to membranes in a partially unfolded molten globule state that is relevant to the activity of the protein.