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Novel preparation and characterization of the alpha4-loop-alpha5 membrane-perturbing peptide from the Bacillus
Somphob Leetachewa1, Gerd Katzenmeier, Chanan Angsuthanasombat
1Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Nakornpathom, Thailand.
Abstract:
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.
Insights
The alpha4-alpha5 hairpin of Bacillus thuringiensis Cry4Ba toxin is crucial for mosquito larvicidal activity. This study isolated and characterized this peptide, confirming its role in membrane pore formation.
Area of Science:
- Molecular Biology
- Biochemistry
- Toxicology
Background:
- Helices 4 and 5 of Bacillus thuringiensis Cry4Ba delta-endotoxin are implicated in mosquito larvicidal activity and membrane pore formation.
- An efficient strategy was developed to produce the alpha4-alpha5 hairpin peptide.
Purpose of the Study:
- To produce and characterize the alpha4-loop-alpha5 hairpin peptide from Cry4Ba toxin.
- To investigate the role of this hairpin in membrane perturbation and pore formation.
Main Methods:
- Engineered a Cry4Ba mutant protein with an additional tryptic cleavage site.
- Expressed and solubilized inclusions in Escherichia coli, followed by trypsin digestion.
- Purified the 7-kDa alpha4-loop-alpha5 hairpin using size-exclusion FPLC and reversed-phase HPLC.
- Analyzed secondary structure via circular dichroism spectroscopy.
- Assessed membrane perturbation using fluorimetric calcein-release assays.
Main Results:
- A 7-kDa fragment, identified as the alpha4-loop-alpha5 hairpin, was efficiently produced and purified.
- The 7-kDa peptide exists predominantly as an alpha-helical structure.
- The helical hairpin demonstrated high activity in perturbing unilamellar liposomes, exceeding that of the full-length activated toxin.
Conclusions:
- The alpha4-loop-alpha5 hairpin is directly involved in the membrane perturbation activity of Cry4Ba toxin.
- These findings provide direct support for the hairpin's role in the pore-forming mechanism of the full-length Cry4Ba toxin.
- The study validates an efficient method for producing functional toxin fragments.
