Rational protein design to improve the thermal stability of a Bacillus thuringiensis Vip3A protein

Vip3A proteins produced by the Bacillus thuringiensis bacterium exhibit insecticidal activity against various crop-damaging lepidopteran pests. However, their limited thermal stability and short shelf life render them unsuitable for use as biopesticides. In this study, we employed rational protein design to enhance the thermal stability of Vip3A64 while minimizing its negative impact on insecticidal activity. This involves substituting potential amino acids based on structural data and using a computational tool (HoTMuSiC) to predict the effect of mutations on the protein's thermal stability as defined by the melting temperature (Tm). We then introduced eight single amino acid substitutions (V239T, V320K, A351C, A351F, D621I, N633Y, E754W, and Q771I) into the tetrameric core and solvent-exposed domains of Vip3Aa64. As determined by the protein thermal shift (PTS) assays, the N633Y and V239T mutants exhibit higher protein melting temperatures than those of the wild type, indicating their enhanced thermal stability. In contrast to other mutants, V239T and N633Y retained their insecticidal activity after one hour of exposure to the high temperature of 55 ˚C. Furthermore, after one month of storage at 37 ˚C, N633Y was the only mutant capable of killing Spodopteraexigua larvae, indicating that it is more stable than the other mutants and the wild type. This study highlights the potential of rational protein design to improve thermal stability and provides a framework for developing effective insecticidal proteins for sustainable agriculture.