Investigation of the unfolding pathway of Bacillus thuringiensis Cyt2Aa2 toxin reveals an unfolding intermediate.
Cyt2Aa2 is a cytolytic toxin from Bacillus thuringiensis subsp. darmstadiensis. Its active form has a lethal activity against specific mosquito larvae. We characterized an unfolding pathway of Cyt2Aa2 using a guanidinium hydrochloride denaturation. The results revealed three-state transition with a detectable intermediate in a condition with 3-4M of GuHCl. The conformational free energies for native and intermediate state unfolding were 5.82+/-0.47 and 16.85+/-1.47kcal/mol, respectively. Kinetic analysis suggested that the activation energy of both transitions was around 23-25kcal/mol, with a rate-limiting step in the second transition. These results have established an energy profile of the Cyt2Aa2 toxin in various conformations involved in the unfolding/refolding pathway. Further characterization of the intermediate state by dye-binding assay, intrinsic fluorescence, and circular dichroism spectroscopy demonstrated characteristics of a molten globule state. This revealed intermediate could play an active role in the structural folding and biological activity of the toxin.
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhonpathom, Thailand.
Anchanee
A
Sangcharoen
Weerachon
W
Tepanant
Somruathai
S
Kidsanguan
Boonhiang
B
Promdonkoy
Chartchai
C
Krittanai
D001413:Bacillus thuringiensis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001426:Bacterial Proteins; D003603:Cytotoxins; D004731:Endotoxins; D011994:Recombinant Proteins; D019791:Guanidine