title: The transmembrane domain of caveolin-1 exhibits a helix-break-helix structure.
abstract: Caveolin is an integral membrane protein that is found in high abundance in caveolae. Both the N- and C- termini lie on the same side of the membrane, and the transmembrane domain has been postulated to form an unusual intra-membrane horseshoe configuration. To probe the structure of the transmembrane domain, we have prepared a construct of caveolin-1 that encompasses residues 96-136 (the entire intact transmembrane domain). Caveolin-1(96-136) was over-expressed and isotopically labeled in E. coli, purified to homogeneity, and incorporated into lyso-myristoylphosphatidylglycerol micelles. Circular dichroism and NMR spectroscopy reveal that the transmembrane domain of caveolin-1 is primarily α-helical (57-65%). Furthermore, chemical shift indexing reveals that the transmembrane domain has a helix-break-helix structure which could be critical for the formation of the intra-membrane horseshoe conformation predicted for caveolin-1. The break in the helix spans residues 108 to 110, and alanine scanning mutagenesis was carried out to probe the structural significance of these residues. Our results indicate that mutation of glycine 108 to alanine does not disrupt the structure, but mutation of isoleucine 109 and proline 110 to alanine dramatically alters the helix-break-helix structure. To explore the structural determinants further, additional mutagenesis was performed. Glycine 108 can be substituted with other small side chain amino acids (i.e. alanine), leucine 109 can be substituted with other β-branched amino acids (i.e. valine), and proline 110 cannot be substituted without disrupting the helix-break-helix structure.
authors.affiliation: Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA.
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Lee
authors.forename: Kerney Jebrell
authors.initials: KJ
authors.lastname: Glover
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D051242:Caveolin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D008823:Micelles; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D010715:Phosphatidylglycerols / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C495540:CAV1 protein, human; D051242:Caveolin 1; D008823:Micelles; D010715:Phosphatidylglycerols; D011994:Recombinant Proteins