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 1. Block I of cryIIIA delta-endotoxin contains a 23 amino acid amphiphilic alpha-helix termed alpha5. The potential involvement of this alpha5 helix in the toxic mechanism of delta-endotoxin was examined. For this purpose, a peptide corresponding to the alpha5 segment and its proline incorporated analogue (P-alpha5) were synthesized and characterized. The alpha-helical content of the peptides, assessed in methanol by circular dichroism (CD), was 58% and 24% for alpha5 and P-alpha5, respectively. To monitor the interaction of alpha5 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 alpha5 and P-alpha5, and revealed that their N-terminals are located within the lipid bilayers. The shape of the binding isotherms indicated that alpha5 aggregated in both zwitterionic and acidic vesicles. Functional characterization of the alpha5 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. alpha5 was much more active than P-alpha5 in both assays. Moreover, membrane-bound alpha5 was more protected from enzymatic proteolysis than P-alpha5. The results reveal a good correlation between the structure and function of alpha5 and experimentally support the hypothesis that alpha5 is a structural component of the pores formed by delta-endotoxins via aggregation of amphiphilic alpha-helices.