Site-directed mutations of thermostable direct hemolysin from Grimontia hollisae alter its arrhenius effect and biophysical properties

Yu Kuo Wang, Sheng Cih Huang, Yi Fang Wu, Yu Ching Chen, Yen Ling Lin, Manoswini Nayak, Yan Ren Lin, Wen Hung Chen, Yi Rong Chiu, Tien Hsiung li Thomas, Bo Sou Yeh, Tung-Kung Wu*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 °C but is reactivated by additional heating above 80 °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-rTDH WT . Circular dichroism measurements of Gh-rTDH WT and Gh-rTDH mut 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.

Original languageEnglish
Pages (from-to)333-346
Number of pages14
JournalInternational journal of biological sciences
Volume7
Issue number3
DOIs
StatePublished - 1 Jan 2011

Keywords

  • Arrhenius effect
  • Circular Dichroism
  • Grimontia hollisae
  • Thermostable direct hemolysin
  • Virulence factor

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