Participation of valine 171 in alpha-Helix 5 of Bacillus thuringiensis Cry1Ab delta-endotoxin in translocation of toxin into Lymantria dispar midgut membranes

Abstract

The Cry1Ab δ-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar, which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This is an insect-specific mechanism; similar results were not observed in Manduca sexta, another Cry1Ab δ-endotoxin-susceptible insect.

FIG. 1.

(A) Three-dimensional image of Cry1Ab δ-endotoxin created by molecular modeling as described in reference , indicating α-helix 5, Val171, and Leu157. (B) Analysis by SDS-10% PAGE of the Cry1Ab (lane 1), L157C (lane 2), and V171C (lane 3) toxins. Lane 4 shows molecular weight markers (Bio-Rad).

FIG. 2.

(A) Thermal unfolding of Cry1Ab wild-type toxin. The toxin unfolds when the temperature is increased (•). When the temperature is decreased under the same conditions, the toxin remains in the unfolded state (▵). (B) Thermal unfolding of the V171C mutant (T_m = 63.45 ± 2.5°C), the L157C mutant (T_m = 70.40 ± 2.7°C), and wild-type Cry1Ab (T_m, = 70.52 ± 3.0) toxins. AU, absorbance units.

FIG. 3.

Inhibition of short-circuit current, I_sc, determined by voltage clamping. (A) Percentage of I_sc remaining for Cry1Ab and the V171C and V171C-NEM mutants. The dashed line shows the time at which the toxin was added to the Vc chamber. (B) (a) Slope of the inhibition of short current versus time as a measure of the rate of ion channel formation. (b) Lag time, the time needed for the short-circuit current to drop ∼10% from its initial value (3).