Anchanee SangcharoenWeerachon TepanantSomruathai KidsanguanBoonhiang PromdonkoyChartchai KrittanaiMahidol UniversityThailand National Center for Genetic Engineering and Biotechnology2018-09-132018-09-132009-05-20Journal of Biotechnology. Vol.141, No.3-4 (2009), 137-141016816562-s2.0-67349086916https://repository.li.mahidol.ac.th/handle/20.500.14594/27226Cyt2Aa2 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 3-4 M of GuHCl. The conformational free energies for native and intermediate state unfolding were 5.82 ± 0.47 and 16.85 ± 1.47 kcal/mol, respectively. Kinetic analysis suggested that the activation energy of both transitions was around 23-25 kcal/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. © 2009 Elsevier B.V. All rights reserved.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyImmunology and MicrobiologyArticleSCOPUS10.1016/j.jbiotec.2009.03.012