Structural and functional characterization of the alpha 5 segment of Bacillus thuringiensis delta-endotoxin.

One of the most conserved sequences in various delta-endotoxins is the 30 amino acid long block I. Block I of cryIIIA delta-endotoxin contains a 23 amino acid amphiphilic alpha-helix termed alpha 5. The potential involvement of this alpha 5 helix in the toxic mechanism of delta-endotoxin was examined. For this purpose, a peptide corresponding to the alpha 5 segment and its proline incorporated analogue (P-alpha 5) were synthesized and characterized. The alpha-helical content of the peptides, assessed in methanol by circular dichroism (CD), was 58% and 24% for alpha 5 and P-alpha 5, respectively. To monitor the interaction of alpha 5 peptides with phospholipid membranes, they were selectively labeled at their N-terminal amino acids with the fluorescent probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or carboxyfluorescein. Fluorometric studies allowed the calculation of membrane surface partition constants, which were about 10(4) M-1 for both alpha 5 and P-alpha 5, and revealed that their N-terminals are located within the lipid bilayers. The shape of the binding isotherms indicated that alpha 5 aggregated in both zwitterionic and acidic vesicles. Functional characterization of the alpha 5 peptides was determined by assessing their ability to dissipate a diffusion potential from sonicated small unilamellar vesicles (SUV) composed of zwitterionic or acidic phospholipids and to lyse human erythrocytes. alpha 5 was much more active than P-alpha 5 in both assays. Moreover, membrane-bound alpha 5 was more protected from enzymatic proteolysis than P-alpha 5.(ABSTRACT TRUNCATED AT 250 WORDS)