標題: Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties
作者: Wang, Yu-Kuo
Huang, Sheng-Cih
Wu, Yi-Fang
Chen, Yu-Ching
Lin, Yen-Ling
Nayak, Manoswini
Lin, Yan Ren
Chen, Wen-Hung
Chiu, Yi-Rong
Li, Thomas Tien-Hsiung
Yeh, Bo-Sou
Wu, Tung-Kung
生物科技學系
Department of Biological Science and Technology
關鍵字: Grimontia hollisae;thermostable direct hemolysin;Arrhenius effect;Circular Dichroism;virulence factor
公開日期: 2011
摘要: Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 degrees C but is reactivated by additional heating above 80 degrees C. 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-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/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-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a beta-sheet to alpha-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.
URI: http://hdl.handle.net/11536/26011
ISSN: 1449-2288
期刊: INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES
Volume: 7
Issue: 3
起始頁: 333
結束頁: 346
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