Novel Preparation and Characterization of the α4-loop-α5 Membrane-perturbing Peptide from the Bacillus thuringiensis Cry4Ba δ-endotoxin

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

“…In this study, although the three-dimensional reconstructed model revealed only the propeller-like shape, its reconstructed EM map fitted with a 100-ns MD simulated Cry4Ba structure interacting with an OG micelle could give an idea about the relative positioning of individual domains in the context of the trimeric prepore complex. In line with several previous studies as well as the established mechanistic models for the membrane-bound state of Bt-Cry toxins, a defined hairpin structure of ␣4-loop-␣5 within DI is conceivably required for membrane insertion and pore formation (3,4,9,12). However, the fact that the hydrophobic faces of the outer amphipathic helices of DI face inwards, these three-domain Cry toxins must go through conformational changes, particularly within DI, to convert this pore-forming domain into a transmembrane pore in which the hydrophobic surfaces would be in close contact with the membrane lipids (3,9).…”

“…Of particular interest, we have strengthened the proposed "umbrella-like" mechanistic model (2,11) by providing direct evidence for liposomal membrane-perturbing activity of the purified Cry4Ba pore-forming fragment, i.e. ␣4-loop-␣5 hairpin (12). Additionally, one highly conserved residue, Asn 183 , located in the middle of the transmembrane ␣5, has been found to play an important role in Cry4Ba toxicity and is essentially involved in toxin-pore oligomerization (13).…”

“…Membrane Perturbation Assays-Calcein-entrapped large unilamellar vesicles (LUVs, ϳ80 nm in mean diameter) were prepared from a lipid mixture (Avanti Polar Lipid) of phos-phatidylcholine, phosphatidylethanolamine, and cholesterol (10:10:1 w/w) with 60 mM calcein in carbonate buffer (pH 9.0) using the extrusion method as described previously (12). Suspension concentrations of calcein-entrapped LUVs were estimated by measuring the lipid phosphorus content (27), and a final concentration of 2.5 M was used for the fluorescence dye-release assays.…”

Mosquito Resistance to Bacterial Larvicidal Toxins

“…Our group has also presented direct proof that the Cry4Ba 4-loop-5 hairpin is capable of perturbing the membrane integrity of lipid vesicles, supporting its role as a fundamental membraneinserted pore-forming determinant that could be separated as an isolated helical hairpin retaining at least its functionality (Leetachewa JBMB 2006) [45]. Other membrane permeation studies with synthetic peptides corresponding to Cry1Ac-domain I helices have also demonstrated that the loop connecting 4 and 5 is needed for efficient penetration of these two transmembrane helices into the lipid bilayers to form lytic pores (Gerber JBC 2000) [46].…”

Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

“…Circular dichroism (CD) spectrum of the purified protein (0.45 mg/ml) prepared in the carbonate buffer, pH 10, was measured using a Jasco J-715 CD spectropolarimeter (Jasco Inc., USA) as described earlier (Leetachewa et al, 2006). CD spectra were recorded by using a 1 nm spectral bandwidth for three scans at a rate 20 nm/min and all spectra were subtracted from a baseline.…”