Interactions of caveolin-1 scaffolding and intramembrane regions containing a CRAC motif with cholesterol in lipid bilayers.

Caveolin-1 is a major structural protein of caveolae and specifically binds cholesterol (Chol). The caveolin scaffolding domain is thought to be involved in caveolin-Chol interaction through the sequence V94-T-K-Y-W-F-Y-R101, a motif that matches a cholesterol recognition amino-acid consensus (CRAC). In the present work, three CRAC-containing peptides, corresponding to caveolin-1 94-101, 82-101 and 93-126, were tested to study the role of the CRAC motif in the caveolin-Chol interaction in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using differential scanning calorimetry (DSC), fluorescence and circular dichroism (CD). The Y97I substituents of the three peptides and one peptide segment corresponding to caveolin-1 101-126 that excludes the CRAC motif were also tested for comparison. Our results showed the potency of these CRAC-containing peptides in sequestering Chol into domains and the enhanced role of the intramembrane domain and scaffolding domain for the potency. Of the three CRAC-containing peptides, the peptide 93-126 was particularly effective in promoting Chol segregation, while the peptide 82-101 was less potent in promoting the formation of domains than the peptide 93-126, but was more potent than the peptide 94-101. The domain partition of DPPC/Chol bilayers was not observed in the presence of the peptide 101-126, in contrast to the case in the presence of the peptide 93-126 at the same concentrations of peptide and Chol. The potency of the CRAC motif in Chol segregation was lowered by the Y97I mutation. The difference in structure may be a factor that contributes to different effects of these peptides on the distribution of Chol in the lipid membrane.