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

Biochemistry
E AlcantaraD H Dean

Abstract

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...Continue Reading

References

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Citations

Dec 26, 2002·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Aug 7, 2007·The Journal of Membrane Biology·Friederike StumpffHolger Martens
Oct 7, 2004·Applied and Environmental Microbiology·Vincent VachonLuke Masson
Jan 5, 2006·Applied and Environmental Microbiology·Cristopher PadillaAlejandra Bravo
Oct 24, 2007·Scandinavian Journal of Immunology·G G Guerrero, L Moreno-Fierros
Mar 31, 2007·Journal of Biochemistry and Molecular Biology·Kasorn Tiewsiri, Chanan Angsuthanasombat

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