Site-Directed Mutations of Thermostable Direct Hemolysin from <i>Grimontia hollisae</i> Alter Its Arrhenius Effect and Biophysical Properties

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

doi:10.7150/ijbs.7.333

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Int J Biol Sci 2011; 7(3):333-346. doi:10.7150/ijbs.7.333 This issue Cite

Research Paper

Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties

Yu-Kuo Wang^1*, Sheng-Cih Huang^1*, Yi-Fang Wu¹, Yu-Ching Chen¹, Yen-Ling Lin¹, Manoswini Nayak¹, Yan Ren Lin¹, Wen-Hung Chen¹, Yi-Rong Chiu¹, Thomas Tien-Hsiung Li^{2 ✉}, Bo-Sou Yeh³, Tung-Kung Wu^{1 ✉}

1. Department of Biological Science and Technology, National Chiao Tung University, 30068, Hsin-Chu, Taiwan, Republic of China
2. Institute of Biochemistry, National Chung Hsing University, 40227, Taichung, Taiwan, Republic of China
3. Hsin Chu General Hospital, Department of Health, Executive Yuan, Taiwan, Republic of China
* These authors contributed equally to this work.

Citation:

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. https://www.ijbs.com/v07p0333.htm

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Abstract

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-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.

Keywords: Grimontia hollisae, thermostable direct hemolysin, Arrhenius effect, Circular Dichroism, virulence factor

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APA

Wang, Y.K., Huang, S.C., Wu, Y.F., Chen, Y.C., Lin, Y.L., Nayak, M., Lin, Y.R., Chen, W.H., Chiu, Y.R., Li, T.T.H., Yeh, B.S., Wu, T.K. (2011). Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties. International Journal of Biological Sciences, 7(3), 333-346. https://doi.org/10.7150/ijbs.7.333.

ACS

Wang, Y.K.; Huang, S.C.; Wu, Y.F.; Chen, Y.C.; Lin, Y.L.; Nayak, M.; Lin, Y.R.; Chen, W.H.; Chiu, Y.R.; Li, T.T.H.; Yeh, B.S.; Wu, T.K. 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.

NLM

CSE

Wang YK, Huang SC, Wu YF, Chen YC, Lin YL, Nayak M, Lin YR, Chen WH, Chiu YR, Li TTH, Yeh BS, Wu TK. 2011. Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties. Int J Biol Sci. 7(3):333-346.

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