title: Site-directed mutations of thermostable direct hemolysin from Grimontia hollisae alter its arrhenius effect and biophysical properties.
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.
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University, 30068, Hsin-Chu, Taiwan, Republic of China.
authors.forename: Yu-Kuo
authors.initials: YK
authors.lastname: Wang
authors.forename: Sheng-Cih
authors.initials: SC
authors.lastname: Huang
authors.forename: Yi-Fang
authors.initials: YF
authors.lastname: Wu
authors.forename: Yu-Ching
authors.initials: YC
authors.lastname: Chen
authors.forename: Yen-Ling
authors.initials: YL
authors.lastname: Lin
authors.forename: Manoswini
authors.initials: M
authors.lastname: Nayak
authors.forename: Yan Ren
authors.initials: YR
authors.lastname: Lin
authors.forename: Wen-Hung
authors.initials: WH
authors.lastname: Chen
authors.forename: Yi-Rong
authors.initials: YR
authors.lastname: Chiu
authors.forename: Thomas Tien-Hsiung
authors.initials: TT
authors.lastname: Li
authors.forename: Bo-Sou
authors.initials: BS
authors.lastname: Yeh
authors.forename: Tung-Kung
authors.initials: TK
authors.lastname: Wu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular; D003224:Congo Red / Q000737:chemistry / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D016297:Mutagenesis, Site-Directed; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014737:Vibrionaceae / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C068489:thermostable direct hemolysin; D003224:Congo Red