title: Loop sequence dictates the secondary structure of a human membrane protein hairpin.
abstract: Membrane proteins adopt two fundamental types of folds in nature: membranes in all organisms harbor α-helical bundles linked by extramembranous loops of varying length, while β-barrel structures are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Here we report that turn-inducing loop mutations in a transmembrane hairpin induce the conversion of an α-helical hairpin to β-sheet oligomers in membrane environments. On the basis of an observation of a sequence bias toward Pro and Gly in the turns of native β-barrel membrane proteins, we characterized in sodium dodecyl sulfate (SDS) micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers several "hairpin" constructs of cystic fibrosis transmembrane conductance regulator transmembrane segments 3 and 4 (TM3-loop-TM4; loop region being (215)IWELLQASA(223)) in which Pro-Gly residues were either inserted or substituted at several positions. Remarkably, suitable positioning of the Pro-Gly doublet caused the adoption of stable β-sheet structures by several mutants in SDS micelles, as shown by circular dichroism spectroscopy, concurrent with a ladder of discrete oligomers observed via SDS-polyacrylamide gel electrophoresis. Reconstitution of wild-type (WT) TM3/4 into POPC vesicles studied by Trp fluorescence, in conjunction with positional quenchers in brominated phospholipids, indicated a transbilayer position for helical WT TM3/4, but likely a largely surface-embedded conformation for the β-sheet mutant with loop region IWPGELLQASA. To the best of our knowledge, such a complete change in the fold with a minimal number of mutations has not been previously observed for a membrane protein. These facile α-helix to β-sheet conversions highlight the contribution of loops to membrane protein structure.
authors.affiliation: Program in Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto M5G 1X8, Ontario, Canada.
authors.forename: Vincent G
authors.initials: VG
authors.lastname: Nadeau
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
mesh_terms: D000595:Amino Acid Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics*; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010713:Phosphatidylcholines / Q000737:chemistry; D011392:Proline / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D008051:Lipid Bilayers; D008823:Micelles; D010713:Phosphatidylcholines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012967:Sodium Dodecyl Sulfate; D011392:Proline; D005998:Glycine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine