Cysteine Scanning Mutagenesis of 4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

Groupe d'étude des protéines membranaires, Université de Montréal, and Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada; Peking University, Peking, People's Republic of China; and Horticultural Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Quebec, Canada

^* To whom correspondence should be addressed. Email: raynald.laprade{at}umontreal.ca.

	Abstract

Helix 4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the 4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicity and pore-forming ability of the mutated toxins were examined, respectively, with bioassays using neonate Manduca sexta larvae and with a light scattering assay using midgut brush border membrane vesicles isolated from this same species. A majority of these mutants had a considerably reduced toxicity and pore-forming ability. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain a significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.