Novel preparation and characterization of the alpha4-loop-alpha5 membrane-perturbing peptide from the Bacillus thuringiensis Cry4Ba delta-endotoxin.
Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba delta-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the alpha4-alpha5 hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the alpha4-loop-alpha5 hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an alpha-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the alpha4-loop-alpha5 hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.
Leetachewa
Somphob
S
Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Nakornpathom, Thailand.
Katzenmeier
Gerd
G
Angsuthanasombat
Chanan
C
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification; D004731:Endotoxins / Q000737:chemistry* / Q000302:isolation & purification; D006460:Hemolysin Proteins; D007306:Insecticides / Q000737:chemistry / Q000302:isolation & purification*; D007915:Lepidoptera / Q000187:drug effects; D008081:Liposomes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D010539:Permeability / Q000187:drug effects; D018272:Porins; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D008081:Liposomes; D010446:Peptide Fragments; D018272:Porins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis