X-ray Serial Synchrotron Crystallography Study of SARS-CoV-2 Main Protease

The main protease (Mpro) is a crucial enzyme needed for the correct functioning of all coronaviruses' lifecycle, it is responsible for the maturation of the majority of viral non- structural proteins (NSP) and has been confirmed to be a valid drug target to halt the viral infection. Currently Mpro is the primary focus for the development of effective therapeutics against the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), since it has a highly conserved structure among variants and it is less prone to induce drug's off-target effects due to the absence of analogue proteases in the human genome with similar specificity. High-quality structural informations are essential for an efficient drug design, these include high resolution structures of protein inhibitors complex for studying drug to target interactions. It is also important to have a good fundamental knowledge of the protein biochemical interaction and mechanism, one strategy to collect this type of information is the crystallographic method. Serial crystallography (SX) is a newly emerging technique originally developed to use the femtosecond pulses of X-ray free electron lasers (XFEL) facilities. Here data collection from multiple microcrystals was necessary instead of the classical one crystal experiment due to the high energy transmitted. This technique is being now adopted by more traditional synchrotron sources making it available to the greater scientific community. One of the major advantages brought by SX is the significant reduction in radiation damage present in the crystals, enabling thus a more faithful room-temperature data collection. A second advantage of SX is the possibility to explore time-resolved experiments via a pump-probe set-up limited only by the X-ray pulse length. In this thesis SARS-CoV-2 Mpro was studied at the new serial crystallography beamline ID29 at the European synchrotron radiation facility. Two proteins constructs were used, the active wild type form (WT) and the double mutant form (DM) with inhibited enzymatic activity. These two proteins were successfully expressed, purified and microcrystallized forming thin plate-like shape crystal. Both apo structures were obtained using sheet-on-sheet chips (SOS chip) and tape-drive set-up available at the beamline. Data collection showed the presence of preferred orientations of the crystals during the SOS chips experiment, this was reflected by the anisotropy of the final dataset. This orientation bias of the microcrystals was barely present for the tape-drive experiment which allowed a more homogenius sampling of the reciprocal space. Soaking of the natural substrate NSP4/5 peptide showed the diffusion of the molecule in Mpro DM at sub-millimolar concentration. Time-resolved experiments carried out by mixing the peptide with the microcrystals weren't able to observe the peptide diffusion inside Mpro DM in the 0.5 to 20 seconds range and no reaction was observed in Mpro WT microcrystals in the 30 seconds to 10 minutes range.