| Summary: | 碩士 === 國立交通大學 === 生物科技學系 === 100 === Thermostable direct hemolysin (TDH), a pore forming toxin, has been recognized as a virulent factor in Vibrio parahaemolyticus. It exists as a homo-tetramer in solution. The homologous TDH from Grimontia hollisae shows 87% identity to Vp-TDH, but displays a distinct interface structure between protomers. Notably, the Gh-TDH and equinatoxin II (Eqt II) from sea anemone exhibit a highly structural similarity, whereas Eqt II has been shown to interact with the lipid membrane via specific regions. In order to understand the roles of functional residues that located in the distinct protomer-protomer interface between Gh-TDH and Vp-TDH and to figure out the critical amino acids involved in the putative membrane binding region, a series of biophysical studies on various mutants of TDH were carried out. The protomer-protomer interface related Gh-TDHY53D and Gh-TDHF159D mutants formed dimer conformation, as observed from the Native PAGE. Furthermore, the Gh-TDHY53D and Gh-TDHF159D mutants exhibited slightly lower hemolytic activities as compared to that of Gh-TDHWT. Thus, the Gh-TDH proteins might form dimer-based tetramer and display variant extent of hemolytic activities, depending on different conformational structures. In parallel, no hemolytic activity was observed from the Gh-TDHW39A, Gh-TDHW65A, Gh-TDHY87A, Gh-TDHY107A and Gh-TDHY87A/Y107A mutants. Moreover, neither fluorescent signal on the cell surface nor fluorescence intensity in flow cytometry analysis was observed while Gh-TDHW65A and Gh-TDHY87A conjugated with FITC was treated with erythrocytes or HeLa cells, indicating that the Trp-65 and Tyr-87 might play critical roles in the process of Gh-TDH initial binding membrane. Interestingly, the flow cytometry analysis and cell experiments indicated that Gh-TDHW39A and Gh-TDHY107A possessed the abilities to bind with cell membrane as that of Gh-TDHWT, in spite of the loss of their hemolytic activity. These results indicated that the Trp-39 and Tyr-107 may assist in the post-binding process of Gh-TDH during its hemolytic process. In summary, both the Tyr-53 and Phe-159 showed their importance in directing protein quaternary structure formation. The Trp-65 and Tyr-87 residues might play a role participating in the protein-membranes binding whereas Trp-39 and Tyr-107 were related to the post-binding process in Gh-TDH hemolytic process.
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