Int J Biol Sci 2011; 7(3):333-346. doi:10.7150/ijbs.7.333
Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties
1. Department of Biological Science and Technology, National Chiao Tung University, 30068, Hsin-Chu, Taiwan, Republic of China
Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 oC but is reactivated by additional heating above 80 oC. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDHWT) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDHY53H/T59I and Gh-rTDHT59I/S63T double-mutants and the Gh-rTDHY53H/T59I/S63T triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDHWT. Circular dichroism measurements of Gh-rTDHWT and Gh-rTDHmut showed a conspicuous change from a β-sheet to α-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.
Keywords: Grimontia hollisae, thermostable direct hemolysin, Arrhenius effect, Circular Dichroism, virulence factor
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How to cite this article:
Wang YK, Huang SC, Wu YF, Chen YC, Lin YL, Nayak M, Lin YR, Chen WH, Chiu YR, Li TTH, Yeh BS, Wu TK. Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties. Int J Biol Sci 2011; 7(3):333-346. doi:10.7150/ijbs.7.333. Available from http://www.ijbs.com/v07p0333.htm