title: De novo design, synthesis, and characterization of a pore-forming small globular protein and its insertion into lipid bilayers.
abstract: The question of how to design a water-soluble globular protein remains. We report here the synthesis of a native-like and pore-forming small globular protein (SGP, 69 amino acid residues). The protein was designed to have four helices: a Trp-containing short hydrophobic helix in the middle surrounded by three Tyr-containing long basic amphiphilic helices. Size-exclusion chromatography and CD measurements indicated that in buffer solution SGP is monomeric with a 50% helical structure. SGP did not completely denature even at high temperature (90 degrees C) and at relatively high Gu x HCl concentration so that the denaturant concentration at the midpoint of the transition is 5 M. Dye binding studies and fluorescence energy transfer experiments showed that SGP possesses a hydrophobic binding site and its Trp of the central helix is present at a relatively hydrophobic region and accepts the energy from Tyr(s) in other amphiphilic helices, indicating that SGP takes a stable globular-like structure in aqueous solution. From the depth-dependent fluorescent studies using egg PC liposomes containing n-doxyl fatty acids and brominated phospholipid as quenchers, it was found that the hydrophobic central alpha-helix is able to enter spontaneously into the lipid bilayers and the Trp in the central alpha-helix is located at about the middle of the alkyl chain in the outer layer of the phospholipid bilayer. The peptide is also able to increase the membrane permeability with two modes of current (basal current and single ion channel) in planar phospholipid bilayers, indicating the spontaneous insertion of the protein into the lipid bilayer (basal current) and then the formation of a uniform size of channel pore (14 pS). SGP is useful as a basic and starting model to find good amino acid sequences that fold to a desired protein structure and to search translocation mechanisms from aqueous solution into lipid bilayers.
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Lee
authors.forename: T
authors.initials: T
authors.lastname: Kiyota
authors.forename: T
authors.initials: T
authors.lastname: Kunitake
authors.forename: E
authors.initials: E
authors.lastname: Matsumoto
authors.forename: S
authors.initials: S
authors.lastname: Yamashita
authors.forename: K
authors.initials: K
authors.lastname: Anzai
authors.forename: G
authors.initials: G
authors.lastname: Sugihara
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D007473:Ion Channels / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D010455:Peptides; D011506:Proteins