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

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.