Helix α4 of the Bacillus thuringiensis Cry1Aa toxin plays a critical role in the postbinding steps of pore formation

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DOIResolve DOI: http://doi.org/10.1128/AEM.01930-08
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TypeArticle
Journal titleApplied and Environmental Microbiology
Volume75
Issue2
Pages359365; # of pages: 7
SubjectBacillus thuringiensis; bio; Protein; activity; Circular Dichroism; Dithiothreitol; Permeability; Manduca; microbiology; Biotechnology
AbstractHelix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins’ mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein’s pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.
Publication date
LanguageEnglish
AffiliationNRC Institute for Fuel Cell Innovation (IFCI-IIPC); NRC Biotechnology Research Institute (BRI-IRB); National Research Council Canada
Peer reviewedNo
NRC number52725
NPARC number12423354
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Record identifier2d6687fe-2a09-4967-96f5-3db50f384d75
Record created2009-10-26
Record modified2016-05-09
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