Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis.
To investigate the membrane pore structure of Cyt2Aa1 toxin from Bacillus thuringiensis, 14 single-cysteine substitutions of the toxin were constructed. Five of these mutants (L172C, V186C, L189C, E214C and L220C) yielded characteristic products when processed by proteinase K; other mutants were degraded by this enzyme. Mutants that yielded characteristic proteolysed products and wild-type toxin were labelled with polarity-sensitive acrylodan (6-acryloyl-2-dimethylaminonaphthalene) at the thiol group of cysteine residues. A green-blue shift in the emission spectra was observed with all labelled toxins on transfer from an aqueous solution into a solution containing membranes or liposomes from red blood cells. These results suggested that the label moved into the hydrophobic environment of the membrane or became buried within hydrophobic regions of the protein oligomers. Digestion of membrane-bound labelled toxin with proteinase K did not cause a significant decrease in emission intensity from any of the labelled mutants. This suggests that L172C, V186C, L189C, E214C and L220C are inserted into the membrane and are therefore protected from proteolysis. In contrast, a marked decrease in emission intensity was observed when membrane-bound labelled wild-type toxin was digested with proteinase K. This suggests that Cys-19 does not insert into the membrane. Fluorimetric analysis of delipidated pore complexes suggests that L172C, V186C, L189C and E214C point towards the lipid in the membrane, whereas L220C is either within the hydrophobic environment of the protein oligomers or exposed to the membrane lipids. Most of the Cys-19 from wild-type molecules is enclosed within the hydrophobic pockets of the protein oligomers.
Promdonkoy
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Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, U.K.
Ellar
D J
DJ
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D001413:Bacillus thuringiensis / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers; D006461:Hemolysis / Q000187:drug effects; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational
D001427:Bacterial Toxins; D017931:DNA Primers; C038518:acrylodan; D015081:2-Naphthylamine