Folding and unfolding of the protoxin from Bacillus thuringiensis: evidence that the toxic moiety is present in an active conformation.
The action of trypsin or papain on the 130-kDa crystal protein (protoxin) from Bacillus thuringiensis subsp. kurstaki HD-73 yields a 67-kDa proteinase-resistant toxic fragment (toxin) which is derived from the N-terminal half of the molecule. Sensitivity to proteolysis and fluorescence emission spectroscopy showed that the toxin unfolded to a much greater extent in 6 M guanidinium chloride (GuHCl) than in 8 M urea. Protoxin also unfolded extensively in 6 M GuHCl, whereas in 8 M urea only the C-terminal half of the molecule had unfolded extensively. Both unfolded protoxin and unfolded toxin refolded to their native and biologically active conformations. The biphasic unfolding observed for protoxin suggests that the C-terminal half of the molecule unfolded rapidly, whereas the N-terminal toxic moiety unfolded at a much slower rate, similar to that of the free 67-kDa toxin. A 67-kDa fragment, derived from the N-terminal half of the molecule, could be generated from the protoxin in the presence of either urea or GuHCl by treatment with proteinases. Compared to toxin in denaturants, this fragment was found to be more sensitive to proteolysis. However, on removal of the denaturants the fragment had the same proteinase resistance and cytolytic activity as native toxin. The increased proteinase sensitivity of the fragment generated in the presence of denaturants appears to be due to a perturbation in the conformation of the N-terminal toxic moiety. This perturbation is attributed to the unfolding of the C-terminal region of the protoxin prior to its proteolysis to yield the 67-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
Choma
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Department of Chemistry, University of Ottawa, Ontario, Canada.
Kaplan
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D001413:Bacillus thuringiensis / Q000472:pathogenicity*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D001427:Bacterial Toxins / Q000737:chemistry*; D001681:Biological Assay; D004731:Endotoxins; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006460:Hemolysin Proteins; D006863:Hydrogen-Ion Concentration; D010206:Papain / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D011498:Protein Precursors / Q000633:toxicity* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology; D014508:Urea / Q000737:chemistry
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006146:Guanidines; D006460:Hemolysin Proteins; D011498:Protein Precursors; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014508:Urea; D014357:Trypsin; D010206:Papain; D019791:Guanidine