Role of alpha-helix seven of Bacillus thuringiensis Cry1Ab delta-endotoxin in membrane insertion, structural stability, and ion channel activity.
Domain I of the Cry1Ab insecticidal toxic protein has seven alpha-helices and is considered to be involved in the ion channel activity. While other alpha-helices, particularly alpha-4 and alpha-5, have been extensively explored, the remaining alpha-helices have been slightly studied. Site-directed mutagenesis was used to generate mutations throughout sequences encoding the alpha-helix 7 to test its role in ion channel function. Every amino acid residue in alpha-helix 7 was mutated to alanine. Most resultant proteins, e.g., D225A, 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 degradation to the above proteases but were 8, 30, and 12 times less toxic to M. sexta, respectively, than the wild-type Cry1Ab. Circular dichroism spectroscopy indicated a very small change in the R228A spectrum, while R224A and E235A display the same spectrum as the wild-type protein. These three mutant proteins showed little differences from Cry1Ab when analyzed by saturation binding and competition binding kinetics with (125)I-labeled toxin or by surface plasmon resonance to M. sexta brush border membrane vesicles. More conservative amino acid substitutions were introduced into alpha-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 Rb+, while R224A and R228A had reduced inhibition of short-circuit current for both ions, when analyzed by voltage clamping of M. sexta midguts.
Alcantara
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EP
Department of Entomology, Biophysics Program, The Ohio State University, Columbus, Ohio 43210, USA.
Alzate
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O
Lee
M K
MK
Curtiss
A
A
Dean
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DH
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D006460:Hemolysin Proteins; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D007814:Larva / Q000187:drug effects / Q000378:metabolism; D018402:Manduca; D008871:Microvilli / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary / Q000235:genetics; D011971:Receptors, Immunologic / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013820:Thermolysin / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007473:Ion Channels; D011971:Receptors, Immunologic; C048071:endotoxin receptor; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D013820:Thermolysin