De novo synthesis of a small globular protein SGP and its insertion into lipid bilay

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The question of how to design a water-soluble globular protein remains. We report here a making 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 a middle surrounded by three Tyr-containing long basic amphiphilic helices. Size exclusion chromatography and CD measurement indicated that in buffer solution SGP is monomeric with a 50% helical structure. SGP did not completely denature even at high temperature (90ﾟC) ard at relatively high Gu-Cl concentration that the denaturant concentration at the midpoint of transition is 5M.Dye-binding studies and fluorescence energy transfer experiments showed that SGP possesses a hydrophobic binding site and its Trp of central helix is present at 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 bilayrs and the Trp in central alpha-helix is located at about the middle of the alkyl chain in the outer layr of the phospholipid bilayr. The peptide is also able to increase the membrane permeability with two modes of current (basal current and single ion channel) in planar phospholipid bilayrs, indicating that the spontaneous insertion of the protein into lipid bilayr (basal current) and then the formation of a uniform size of channel pore (14pS). 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 bilayrs.