Role of α-Helix Seven of Bacillus thuringiensis Cry1Ab δ-Endotoxin in Membrane Insertion, Structural Stability, and Ion Channel Activity
| Autor: | Donald H. Dean, Edwin P. Alcantara, and April Curtiss, Mi K. Lee, Oscar Alzate |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Patch-Clamp Techniques
Bacterial Toxins Bacillus thuringiensis Thermolysin Biochemistry Ion Channels Protein Structure Secondary Hemolysin Proteins Protein structure Bacterial Proteins Manduca medicine Animals Intestinal Mucosa Receptors Immunologic Ion channel Alanine chemistry.chemical_classification Bacillus thuringiensis Toxins Microvilli biology Circular Dichroism Hydrolysis Cell Membrane fungi Surface Plasmon Resonance Trypsin biology.organism_classification Amino acid Endotoxins Intestines Amino Acid Substitution chemistry Manduca sexta Larva Mutagenesis Site-Directed Alpha helix Delta endotoxin medicine.drug |
| Zdroj: | Biochemistry. 40:2540-2547 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi0022240 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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