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.

Comprehensive Development Program of Hunter-Killer Peptides for Prostate Cancer

“…We evaluated the efficacy and specificity of CNGRC-GG-D (KLAKLAK) 2 in KS1767 cells, derived from Kaposi sarcoma 26,27 ( Fig. 3a-d Fig.…”

“…During angiogenesis, capillary endothelial cells proliferate and migrate 1,2 . Cord formation is a type of migration that can be studied in vitro by a change in endothelial cell morphology from the usual 'cobblestones' to chains or cords of cells 27 . We tested the effect of CNGRC-GG-D (KLAKLAK) 2 on normal human dermal microvessel endothelial cells (DMECs) in the angiogenic conditions of proliferation and cord formation and in the angiostatic condition of a monolayer maintained at 100% confluency.…”

“…la,b) 27,28 . It is monomeric in solution, and is denatured in a highly cooperative manner, characteristic of native globular-like proteins.…”

“…SGP was designed to mimic this insertion mechanism (Fig. lc) 27,28 . The transition was confirmed in synthetic bilayers, where SGP formed a uniform size pore (14pS) 2…”

Structural Design and Characterization of a Channel-forming Peptide

“…The general concept of de novo design involves the planning and construction of a protein with expectation of folding the linear sequence into a defined three dimensional structure. It has been demonstrated by an increasing number of studies that successful design leads led to the production of proteins and peptides with precise structures and functions (Lee et al, 1997;Wierzbicki et al, 2000;Lear et al, 2001;Schnepf et al, 2001;Wei et al, 2003). Over the past few years, studies and characterizations of protein structures, especially those with a helical conformation have significantly improved our understanding of how secondary structures are formed and stabilized (Gromiha and Selvaraj, 2001;.…”