In silico and in vitro investigations on cry4a and cry11a toxins of Bacillus thuringiensis var israelensis

Document Type: Research Paper

Authors

Department of Genetics, Bhagwan Mahavir Hospital and Research Center, Hyderabad, A.P, India.

Abstract

In the present study we attempted to correlate the structure and function of the cry11a (72 kDa) and cry4a (135 kDa) proteins of Bacillus thuringiensis var israelensis. Homology modeling and secondary structure predictions were done to locate most probable regions for finding helices or strands in these proteins. The JPRED (JPRED consensus secondary structure prediction server) secondary structure predictions were chosen for its ability to predict with high accuracies. The homology model predicted by CPH (CPH Homology Modelling server) modeler showed a distinct region of helices and sheets. The membrane spanning helices were
predicted using TOPPRED (TOPPRED Topology prediction of membrane proteins server); these helices are known to play crucial role in cell lyses. The role of one such segment corresponding to amino acids 132-150 of cry4a protein, which had a large hydrophobic moment, was elucidated. The Circular Dichroism spectra of the peptide showed helical structure in methanol and β sheet structure in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The biological activity of this peptide was investigated. The peptide
showed weak hemolytic activity in vitro. This may be due to the synthetic peptide used rather than the whole molecule in the native environment. The ability of the peptide and the alkali solubilized crystal proteins to perturb the synthetic membrane was investigated using carboxyflourescein trapped liposomes. The leakage caused by alkali solubilized extract was double than the leakage caused by synthetic peptide. In case of alkali solubilized extract, various osmoprotectants were seen to delay lytic activity. Thus it is clear that the cry proteins are highly active and lethal in their native state. Not only the membrane spanning segments but
the whole molecule plays a crucial role in lysis.

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