Effects of pH on comformational properties related to the toxicity of Bacillus thuringiensis δ-endotoxin

Venugopal, M.; Wolfersberger, Michael G.; Wallace, B.A.

doi:10.1016/0167-4838(92)90024-8

Cited by 17 publications

(9 citation statements)

References 28 publications

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“…Venugopal et al have shown that a Cry1 δ‐endotoxin from B . thuringiensis ssp kurstaki undergoes a loss of helical conformation at pH > 10 23. Previous studies with the homolog Cry3A24 as well as this protein (J. Bryson, unpublished results) have shown that under these high pH conditions, the protein exists as a dimer in solution suggesting that large changes in secondary structure are not observed at higher pH for the Cry3 family of toxins.…”

Section: Methodsmentioning

confidence: 87%

The role of a conserved histidine–tyrosine interhelical interaction in the ion channel domain of δ‐endotoxins fromBacillus thuringiensis

Seale

2005

Proteins

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The delta-endotoxin proteins are produced by Bacillus thuringiensis during the sporulation phase of its life cycle. These proteins exhibit insecticidal activity through receptor-mediated ion channel formation. The mode of action of these proteins requires the conversion of the protein from a water-soluble conformation to a membrane-inserted conformation. While there is X-ray structure information for the soluble protein, no detailed structure exists for the membrane-inserted protein. However, based on peptide studies, an umbrella model for the membrane-inserted state has been proposed. Here, we investigated the role of a conserved hydrogen bond interaction between two helices that are suggested to undergo a large conformational change upon membrane insertion. Mutation of either the histidine or the tyrosine resulted in a protein that has significantly reduced bioactivity, increased overall flexibility, and significantly reduced stability. These data highlight an important role for this interaction in the overall stability of the protein. Additionally, the conservation of histidine and tyrosine in these positions may suggest a functional role for the interaction in the conformational switching from soluble to membrane protein.

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Section: Methodsmentioning

confidence: 87%

The role of a conserved histidine–tyrosine interhelical interaction in the ion channel domain of δ‐endotoxins fromBacillus thuringiensis

Seale

2005

Proteins

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“…As electrostatic interactions are important for protein folding and intermolecular interactions, alkaline pH may influence protein structure and macromolecular association (Fujita et al 1998). However, several studies that allowed structural comparison of proteins at different pH indicate that pH 10.0 is not likely to significantly perturb protein structure (Dixon et al 1991; Harries et al 2004; Venugopal et al 1992). The results presented here also do not indicate that protein unfolding at pH 10.0 is a common phenomenon.…”

Section: Resultsmentioning

confidence: 99%

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

et al. 2009

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Summary Reductive methylation of lysine residues in proteins offers a way to introduce 13C methyl groups into otherwise unlabeled molecules. The 13C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of 13C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca2+-bound Calmodulin, Lysozyme, Ca2+-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of 13C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment.

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“…aegypti larvae and they showed that only the inhibition of V-ATPases induced a strong acidification of the midgut pH [62]. As pH affects numerous aspects of toxin action like Bt crystal solubility [7], toxin conformation [63,64], gut enzymes activity [65] and pore formation [66,67], an alteration of gut pH could have a general effect on reducing Bti toxicity. Comparing internal larval midgut pH between resistant and susceptible strains will allow to confirm/infirm if the observed ATPases decreased expression induce an acidification of the gut lumen.…”

Section: Discussionmentioning

confidence: 99%

Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches

et al. 2012

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BackgroundBacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis).ResultsSeveral Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut.ConclusionsBy combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes.

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Effects of pH on comformational properties related to the toxicity of Bacillus thuringiensis δ-endotoxin

Cited by 17 publications

References 28 publications

The role of a conserved histidine–tyrosine interhelical interaction in the ion channel domain of δ‐endotoxins fromBacillus thuringiensis

The role of a conserved histidine–tyrosine interhelical interaction in the ion channel domain of δ‐endotoxins fromBacillus thuringiensis

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches

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