Structural characterization, membrane interaction, and specific assembly within phospholipid membranes of hydrophobic segments from Bacillus thuringiensis var. israelensis cytolytic toxin

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Abstract
The Bacillus thuringiensis var. israelensis (Bti) cytolytic toxin is hypothesized to exert its toxic activity via pore formation in the cell membrane as a result of the aggregation of several monomers. To gain insight into the toxin's mode of action, 2 putative hydrophobic 22 amino acid peptides were synthesized and characterized spectroscopically and functionally. One peptide corresponded to the putative amphiphilic alpha-helical region (amino acids 110-131, termed helix-2), and the other to amino acids 50-71 (termed helix-1) [Ward, E. S., Ellar, D. J., & Chilcott, C. N. (1988) J. Mol. Biol. 202, 527-535] of the toxin. Circular dichroism spectroscopy revealed that both segments adopt high alpha-helical content in a hydrophobic environment, in agreement with previous models. To monitor peptide-lipid and peptide-peptide interactions, the peptides were labeled selectively with either 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) (to serve as donor) or tetramethylrhodamine (to serve as an acceptor), at their N-terminal amino acids. Both segments bind strongly to small unilamellar vesicles, composed of zwitterionic phospholipids, with surface partition coefficients on the order of 10(4) M-1. The shape of the binding isotherms indicates that helix-2 forms large aggregates within phospholipid membranes. Resonance energy transfer experiments demonstrated that the segments self-associate and interact with each other, but do not associate with unrelated membrane-bound peptides. Functional characterization demonstrated that helix-2 permeates phospholipid SUV with a potency similar to that of naturally occurring pore-forming peptides. Thus, the results support a role for helices-1 and -2 in the assembly and in the pore formation by Bti toxin.