The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.
Spinach thylakoids were immobilized onto multiwalled carbon nanotubes using a molecular tethering chemistry. The resulting thylakoid-carbon nanotube composites showed high photo-electrochemical activity under illumination. Multiple membrane proteins have been observed to participate in direct electron transfer with the electrode, resulting in the generation of photocurrents, the first of its kind reported for natural photosynthetic systems. Upon inclusion of a mediator, the photo-activity was enhanced. The major contributor to the photocurrent was the light-induced water oxidation reaction at the photosystem II complex. The thylakoid-MWNT composite electrode yielded a maximum current density of 68 mA cm À2 and a steady state current density of 38 mA cm À2 , which are two orders of magnitude larger than previously reported for similar systems. The high electrochemical activity of the thylakoid-MWNT composites has significant implications for both photosynthetic energy conversion and photofuel production applications. A fuel cell type photosynthetic electrochemical cell developed using a thylakoid-MWNT composite anode and laccase cathode produced a maximum power density of 5.3 mW cm À2 , comparable to that of enzymatic fuel cells. The carbon based nanostructured electrode has the potential to serve as an excellent immobilization support for photosynthetic electrochemistry based on the molecular tethering approach as demonstrated in this work. Broader contextThe article reports a photosynthetic electrochemical cell composed of a thylakoid-based photo-anode and laccase based enzymatic cathode. The cell generates electricity based on photo-induced water oxidation at the cathode and enzyme catalyzed oxygen reduction at the cathode under neutral pH conditions. The thylakoids and laccase were molecularly tethered to carbon nanotube modied electrodes using hetero bi-functional cross-linkers. For the rst time in natural photosynthetic systems, multiple membrane proteins have been shown to participate in direct electron transfer with the nanostructured electrode. The origin of photo-activity was conrmed to be from the light induced water-splitting reaction at the oxygen-evolving site of thylakoids. The thylakoid-MWNT composite electrode yielded a maximum current density of 68 mA cm À2 and a steady state current density of 38 mA cm À2 , which are two orders of magnitude larger than those previously reported for similar systems. The direct conversion of light into electricity using plant thylakoids demonstrated in this work offers great potential for green energy harvesting. The high photo-electrochemical activity of thylakoids reported here has potential implications in photofuel production and other articial photosynthesis applications.
The main protease (3CL Mpro) from SARS-CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL Mpro possesses an unusual catalytic dyad composed of Cys145 and His41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL Mpro, which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form where Cys145 is in the negatively charged thiolate state, and His41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen bonding networks in the active site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme’s electrostatic environment.
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