Caveolin-1 hydrophobic segment peptides insertion into membrane mimetic systems: Role of Proline residue

Abstract

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.

Structural determinants of protein partitioning into ordered membrane domains and lipid rafts

“…Further, GUVs can be manipulated after formation to produce highly curved tubules, for example to study the effects of membrane curvature on protein binding (Roux, Cuvelier et al 2005, Tian and Baumgart 2009, Aimon, Callan-Jones et al 2014. Smaller vesicles have been probed by X-ray scattering to determine conformational changes upon membrane binding (Lee, Lee et al 2014), electron microscopy to study membrane tubulation by curvature generating proteins (Shi and Baumgart 2015) or molecular motors (Roux, Cappello et al 2002), and circular dichroism to study the effects of membranes on protein secondary structure (Aoki and Epand 2012). An important technique for evaluation of partitioning in LUVs is Förster Resonance Energy Transfer (FRET), which measures the molecular proximity (interpreted as co-partitioning) between a protein of interest and a wellcharacterized marker for a particular membrane domain (Lin and London 2013).…”

Reconstitution and spectroscopic analysis of caveolin-1 residues 62–178 reveals that proline 110 governs its structure and solvent exposure

“…Proline 110 is postulated to be a key residue of the intramembrane turn of caveolin-1 [8, 9, 11–13]. Therefore, proline 110 could be essential for the proper fold of Cav1 62–178 , and its mutation to alanine could cause significant changes in the secondary structure of Cav1 62–178 .…”

“…However, this postulation was not supported by an in vitro glycosylation study which concluded that P110A had a topology identical to that of the wild-type protein [10]. In contrast, studies using a short, solubility-enhanced construct encompassing residues 103–122, showed significant changes in bilayer depth and α-helicity when the P110A mutation was made [11]. …”

Recent Progress in the Topology, Structure, and Oligomerization of Caveolin: A Building Block of Caveolae

“…In a study which compared the solvent accessibility of wild-type and P110A IMD constructs (residues 102e122) using fluorescence quenching and l max determinations, it was found that the overall tryptophan environment appeared to differ between constructs (with the P110A mutant becoming less solvent accessible), consistent with a topological change for the mutant (Aoki & Epand, 2012). Additionally, circular dichroism spectroscopy was used to compare the secondary structure of the wild-type and mutant and showed an increase in the a-helical character on the order of 1.5-to 2-fold in the case of the mutant, consistent with the reported linearization of the protein upon P110A mutation.…”