Structural and functional characterization of the .alpha.5 segment of Bacillus thuringiensis .delta.-endotoxin
- 1 April 1993
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 32 (13), 3429-3436
- https://doi.org/10.1021/bi00064a029
Abstract
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)Keywords
This publication has 48 references indexed in Scilit:
- Interaction of D-amino acid incorporated analogs of pardaxin with membranesBiochemistry, 1992
- Isolation, amino acid sequence and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skinBiochemistry, 1991
- Synthesis, location, and lateral mobility of fluorescently labeled ubiquinone 10 in mitochondrial and artificial membranesBiochemistry, 1989
- Interaction between the δ‐endotoxin produced by Bacillus thuringiensis ssp. entomocidus and liposomesFEBS Letters, 1988
- Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipidsBiochemistry, 1987
- Bacillus thuringiensis toxin inhibits K+‐gradient‐dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cellsFEBS Letters, 1986
- The structure of the voltage‐sensitive sodium channelFEBS Letters, 1985
- Differential light scattering and absorption flattening optical effects are minimal in the circular dichroism spectra of small unilamellar vesiclesBiochemistry, 1984
- Diffusion potential cascade. Convenient detection of transferable membrane poresBiochemistry, 1983
- Incorporation of Melittin into phosphatidylcholine bilayersFEBS Letters, 1981