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pH Induces Thermal Unfolding of UTI: An Implication of Reversible and Irreversible Mechanism Based on the Analysis of Thermal Stability, Thermodynamic, Conformational Characterization.

Fan H, Liu J, Ren W, Zheng Z, Zhang Y, Yang X, Li H, Wang X, Zou G

State Key Laboratory of Virology, Life Sciences’ College, Wuhan University, Wuhan, 430072, China.

The thermal unfolding of Urinary Trypsin Inhibitor (UTI) was studied by several methods: Circular Dichroism (CD), Fluorescence and UV-Vis spectra. Thermal melting of UTI, dissolved in the neutral and basic buffers, was proved to be irreversible and two domains of UTI unfolded simultaneously, but the melting was reversible and the intermediate was observed when pH is lower than 4.2. The result suggested that heat and changes in pH, which had a more important impact on the stabilization of the domain I and the interaction between two domains, might cause different unfolding transitions. A reasonable explanation was deduced for the mechanism of reversible and irreversible thermal unfolding based on the effect of pH on the protein structure, the analysis of thermal transitions and the result of Electron Microscopy: In neutral and basic buffers, the Reactive Central Loop (RCL) in domain II can interact with or insert into the partial expanding domain I and UTI become self-polymerization, however, no aggregation can be observed in acid buffer since low pH and heat destabilized the structure of the domain I and the native conformation can restructure. The interaction between the special structural element RCL and domain I play an important role in the formation of polymer which was different from other two reasons given by other authors-the cleavage of disulfide and the formation of irregular polymer mainly based on hydrophobic interaction.

Published 11 March 2008 in J Fluoresc, 18(2): 305-317.
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