| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 100 === The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus is essential to viral replication. Besides proteolytic activity, it was proved that PLpro also processes deubiquiting and de-ISGylating activities. Here, I illustrated that the PLpro-(1600-1858) fragment, which contains the thumb, palm, and fingers domain, is capable for catalysis. The unfolding of PLpro in urea was measured to delineate the structural stability and unfolding character. By circular dichroism, the unfolding of the secondary structure of PLpro in various concentrations of urea was a monophasic process with [Urea]0.5, N-U at 4.4 M. The tertiary structural unfolding of PLpro was examined in terms of fluorescence spectroscopy. The average fluorescence emission wavelength by excited at 280 nm in various urea is a biphasic process, and the unfolding intermediates with [Urea]0.5, N-I and [Urea]0.5, I-U at 0.34 and 5.8 M, respectively. Moreover, the tryptophan fluorescence used to measure the unfolding of the catalytic core showed a monophasic process with [Urea]0.5, N-U at 4.7 M. It suggested that the second transition comes from the unfolding of thumb-palm domains. Furthermore, EDTA experiments also indicated that the first transition was due to the unfolding of the zinc-binding domain. The unfolding intermediate of the enzyme thus represented a folded catalytic core but an unfolded zinc-binding domain. In the unfolding process, a molten globule state with exposure of interior hydrophobic area was represented at the beginning of catalytic core unfolding. Furthermore, an 80% lost of its enzymatic activity at urea concentration at 1.1 M showed closely correlated with unfolding of the zinc-binding domain. It indicated that the structure integrity of zinc-binding site is essential for the complete proteolytic and deubiquitinating activity. The unfolding/refolding process and the importance of zinc binding will not only suite to SARS-CoV PLpro, but also delineate to other coronavirus PLpro and deubiquitinating enzymes with similar scaffold.
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