Biophysical Analysis of Vip3Aa Toxin Mutants Before and After Activation

Pongsatorn Khunrach, Wahyu Surya, Boonhiang Promdonkoy, Jaume Torres*, Panadda Boonserm*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Cry toxins from Bacillus thuringiensis are effective biopesticides that kill lepidopteran pests, replacing chemical pesticides that indiscriminately attack both target and non-target organisms. However, resistance in susceptible pests is an emerging problem. B. thuringiensis also produces vegetative insecticidal protein (Vip3A), which can kill insect targets in the same group as Cry toxins but using different host receptors, making the combined application of Cry and Vip3A an exciting possibility. Vip3A toxicity requires the formation of a homotetramer. Hence, screening of Vip3A mutants for increased stability requires orthogonal biophysical assays that can test both tetrameric integrity and monomeric robustness. For this purpose, we have used herein for the first time a combination of analytical ultracentrifugation (AUC), mass photometry (MP), differential static light scattering (DSLS) and differential scanning fluorimetry (DSF) to test five mutants at domains I and II. Although all mutants appeared more stable than the wild type (WT) in DSLS, mutants that showed more dissociation into dimers in MP and AUC experiments also showed earlier thermal unfolding by DSF at domains IV–V. All of the mutants were less toxic than the WT, but toxicity was highest for domain II mutations N242C and F229Y. Activation of the protoxin was complete and resulted in a form with a lower sedimentation coefficient. Future high-resolution structural data may lead to a deeper understanding of the increased stability that will help with rational design while retaining native toxicity.

Original languageEnglish
Article number11970
JournalInternational Journal of Molecular Sciences
Volume25
Issue number22
DOIs
Publication statusPublished - Nov 2024
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2024 by the authors.

ASJC Scopus Subject Areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Keywords

  • Bacillus thuringiensis
  • differential static light scattering
  • insecticidal protein
  • mass photometry
  • sedimentation velocity
  • Vip3A

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