Time-resolved fluorescence studies reveal differences in dynamic motion between main proteases of SARS-CoV-2 and SARS-CoV.

The main protease (M ^pro ) is an attractive drug target for inhibiting the coronavirus. Lots of research has focused on the static viewpoint of the M ^pro , such as the X-ray crystal structure, inhibitors design, and the transition between monomer and dimer. However, the attention to the dynamical features of M ^pro is limited, which is essential for a deeper determination of the properties of the target protein. In our research, we constructed three single-tryptophan mutants (W31IN, W207IN, and W218IN) of M ^pro from SARS-CoV-2 and SARS-CoV to monitor the motion of M ^pro at the nano-second timescale using the time-resolved fluorescence assay. We found that the temperature-dependent Stokes shift results show various behaviors among the three single-tryptophan mutants: the microenvironment around the Trp207 residue is more temperature-sensitive compared to that in residues Trp31 and Trp218. The molecular dynamic simulation results further support that M ^pro _SARS is more flexible than that of M ^pro _SARS2 . This difference is directly related to the distinct perturbations of residues Phe185 to Gln192, a loop that connects domain II and domain III. For the first time, we were able to reveal the different motions between M ^pro _SARS2 and M ^pro _SARS , although the static structures of these two are not distinguished. The differences in dynamics would be an essential step towards understanding the evolving trend of coronavirus, providing a comprehensive view of the properties of M ^pro , and offering perspectives for designing inhibitors for further research.
Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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