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.
Sangcharoen
Anchanee
A
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhonpathom, Thailand.
Tepanant
Weerachon
W
Kidsanguan
Somruathai
S
Promdonkoy
Boonhiang
B
Krittanai
Chartchai
C
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