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.