Effects of disulfide bridges in domain I of Bacillus thuringiensis Cry1Aa delta-endotoxin on ion-channel formation in biological membranes.
The delta-endotoxin family of toxic proteins represents the major component of the insecticidal capability of the bacterium Bacillus thuringiensis. Domain I of the toxins, which is largely alpha-helical, has been proposed to unfold at protein entry into the membrane of a target insect, following models known as the penknife and umbrella models. We extended the analysis of a previous work in which four disulfide bridges were constructed in domain I of the Cry1Aa delta-endotoxin that putatively prevented unfolding during membrane partitioning. Using bioassays and voltage clamping of whole insect midgut instead of artificial lipid bilayers, it was found that, while toxicity and inhibition of the short-circuit current were reduced, only one of the disulfide bridges eliminated the activity of the toxins in the insect midgut membrane, and in that case, the loss of toxicity was due to the single amino acid substitution, R99C. It is proposed that at least alpha helices 4, 5, 6, and 7 and domain II partition in the midgut membranes of target insects, in support of an insertion model in which the whole protein translocates into the midgut membrane.
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Biochemistry Department, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA. alzate@ neuro.duke.edu
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D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D012831:Bombyx; D004220:Disulfides / Q000737:chemistry*; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D007306:Insecticides / Q000737:chemistry*; D007473:Ion Channels / Q000096:biosynthesis*; D018402:Manduca; D018408:Patch-Clamp Techniques; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004220:Disulfides; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D007473:Ion Channels; C030956:insecticidal crystal protein, Bacillus Thuringiensis