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

The main protease (Mpro) is an attractive drug target for inhibiting the coronavirus. Lots of research has focused on the static viewpoint of the Mpro, such as the X-ray crystal structure, inhibitors design, and the transition between monomer and dimer. However, the attention to the dynamical features of Mpro 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 Mpro from SARS-CoV-2 and SARS-CoV to monitor the motion of Mpro 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 MproSARS is more flexible than that of MproSARS2. 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 MproSARS2 and MproSARS, 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 Mpro, and offering perspectives for designing inhibitors for further research. © 2024