Protocol for production and purification of SARS-CoV-2 3CLpro.

3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L-1 following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CLpro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay. For complete details on the use and execution of this protocol, please refer to Bafna et al.1.

G1/S transcription factors assemble in increasing numbers of discrete clusters through G1 phase.

In budding yeast, the transcription factors SBF and MBF activate a large program of gene expression in late G1 phase that underlies commitment to cell division, termed Start. SBF/MBF are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the questions of how SBF/MBF are dynamically distributed across the G1/S regulon and how this impacts the Start transition. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits in fixed cells revealed each transcription factor was organized into discrete clusters containing approximately eight copies regardless of cell size and that the total number of clusters increased as cells grew through G1 phase. Stochastic modeling using reasonable biophysical parameters recapitulated growth-dependent SBF/MBF clustering and predicted TF dynamics that were confirmed in live cell PALM experiments. This spatio-temporal organization of SBF/MBF may help coordinate activation of G1/S regulon and the Start transition.

Hydration of the folding transition state ensemble of a protein.

A complete description of the mechanisms of protein folding requires knowledge of the structural and physical character of the folding transition state ensembles (TSEs). A key question concerning the role of hydration of the hydrophobic core in determining folding mechanisms remains. To address this, we probed the state of hydration of the TSE of staphylococcal nuclease (SNase) by examining the fluorescence-detected pressure-jump relaxation behavior of six SNase variants in which a residue in the hydrophobic core, Val-66, was replaced with polar or ionizable residues (Lys, Arg, His, Asp, Glu, and Asn). Because of a large positive activation volume for folding, the major effect of pressure on the wild-type protein is to decrease the folding rate. By the time wild-type SNase reaches the folding transition state, most water has already been expelled from its hydrophobic core. In contrast, the major effect of pressure on the variant proteins is an increase in the unfolding rate due to a large negative activation volume for unfolding. This results from a significant increase in the level of hydration of the TSE when an internal ionizable group is present. These data confirm that the role of water in the folding reaction can differ from protein to protein and that even a single substitution in a critical position can modulate significantly the properties of the TSE.