Role of alpha-helix seven of Bacillus thuringiensis Cry1Ab delta-endotoxinin membrane insertion, structural stability, and ion channel activity

Domain I of the Cry1Ab insecticidal toxic protein has seven a-helices and i s considered to be involved in the ion channel activity. While other alpha -helices, particularly alpha -4 and alpha -5, have been extensively explore d, the remaining a-helices have been slightly studied. Site-directed mutage nesis was used to generate mutations throughout sequences encoding the alph a -helix 7 to test its role in ion channel function. Every amino acid resid ue in a-helix 7 was mutated to alanine. Most resultant proteins, e.g., D225 A, W226A, Y229A, N230A, R233A, R234A, D242A, and F247A yielded no protoxin or were sensitive to degradation by trypsin or Manduca sexta midgut juice. Other mutant proteins, R224A, R228A, and E235A, were resistant to degradati on to the above proteases but were 8, 30, and 12 times less toxic to M. sex ta, respectively, than the wild-type Cry1Ab. Circular dichroism spectroscop y indicated a very small change in the R228A spectrum, while R224A and E235 A display the same spectrum as the wild-type protein. These three mutant pr oteins showed little differences from Cry1Ab when analyzed by saturation bi nding and competition binding kinetics with I-125-labeled toxin or by surfa ce plasmon resonance to M, sexta brush border membrane vesicles. More conse rvative amino acid substitutions were introduced into ct-helix 7 residues: R228K, F232Y, E235Q, and F247Y. In comparison with wild-type Cry1Ab, mutant proteins R228K, F232Y, E235A, and E235Q selectively discriminate between K + and Rbf, while R224A and R228A had reduced inhibition of short-circuit cu rrent for both ions, when analyzed by voltage clamping of M. sexta midguts.