- Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122 that are anchored to the membrane with cholesterol-rich domains. Previously, we reported that changing the Pro(110) residue to Ala (the P110A mutant) prevents not only the localization of the protein into lipid rafts but also the formation and functioning of caveolae. The conformational state of caveolin-1 can be shifted toward the transmembrane arrangement by this single amino acid mutation. To model the conformation, and extent of membrane insertion of this segment into membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of caveolin-1 having the sequence KKKKLSTIFGIPMALIWGIYFAILKKKKK-amide and the mutated version, KKKKLSTIFGIAMALIWGIYFAILKKKKK-amide. These peptides contain flanking Lys residues to facilitate purification and handling of the peptide. Circular dichroism measurements demonstrated that the mutated peptide has increased helical content compared with the wild type both in the presence and absence of lipid. The fluorescence emission from the Trp residues in the peptide showed significant blue shifts in the presence of liposomes, however the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. Our overall findings support our studies with the intact protein in cells and suggest that the peptide of WT caveolin-1 hydrophobic segment has an intrinsic preference not to maintain its conformation as a rigid transmembrane helix. Substituting the Pro residue with an Ala allows the peptide to exist in a more hydrophobic environment likely as a consequence of a change in its conformation to a straight hydrophobic helix that traverses the membrane.