key: cord-0801089-qeemcx4h authors: Almutairi, Ghada Obeid; Malik, Ajamaluddin; Alonazi, Mona; Khan, Javed Masood; Alhomida, Abdullah S.; Khan, Mohd Shahnawaz; Alenad, Amal M.; Altwaijry, Nojood; Alafaleq, Nouf Omar title: Expression, purification, and biophysical characterization of recombinant MERS-CoV main (M(pro)) protease date: 2022-04-19 journal: Int J Biol Macromol DOI: 10.1016/j.ijbiomac.2022.04.077 sha: 65c3fe0ad4b79c320ab9837b5a9c3ccd22463735 doc_id: 801089 cord_uid: qeemcx4h MERS-CoV main protease (M(pro)) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV M(pro) under different conditions is poorly characterized. In this study, MERS-CoV M(pro) was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV M(pro) tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV M(pro) was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of M(pro) showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV M(pro) forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 M(pro) shows 96.08% and 50.65% similarity to that of SARS-CoV M(pro) and MERS-CoV M(pro), respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV M(pro). Human coronaviruses were first characterized in the 1960s as the causative agents for generally mild to moderate respiratory infections. In 2003, the outbreak of novel human coronavirus causing severe acute respiratory syndrome (SARS) was reported as a global public health threat of the 21 st century, severe-acute respiratory syndrome (SARS), and the virus was termed as SARS-CoV. SARS is an atypical form of pneumonia [1] that occurred in China and spread to 30 countries infecting more than 8000 people with a 10% case fatality rate (CFR) [2] . Another novel coronavirus causing severe acute respiratory syndrome emerged in the Middle East in 2012, with a higher CFR (35%), called Middle East respiratory syndrome (MERS-CoV) [2, 3] . Recently in 2019 an outbreak of SARS-CoV-2 started in Wuhan City, Hubei Province of China, causing unusual viral pneumonia, recognized as coronavirus disease 2019 (COVID-19) [4] . SARS-CoV-2, which causes COVID-19 disease, quickly became pandemic due to direct human-to-human transmissions. As of November 08, 2021, COVID-19 infected over 248 million people and resulted in around 5 million deaths (https://covid19.who.int/). Coronaviruses tend to mutate and infect several hosts. Therefore, they can jump between species and causes outbreaks in human and animals [5] . There are no effective antiviral drugs for human CoV infection [6] . Viral proteases are essential for the maturation of the viral proteins, thus vital for the coronavirus life-cycle [7] . MERS-CoV open-reading frame 1 (ORF1) encodes a viral replication and maturation, making them attractive targets for discovering antiviral drugs [7, 9] . MERS-CoV M pro (3CL pro ) is a chymotrypsin-like cysteine protease, and because of its dominant role in the post-translational processing of the polyprotein, it is more commonly known as main protease (M pro ) [9] . MERS-CoV M pro is a dimeric protein and has a conserved catalytic dyad (Cys148-His41) and an extended binding site consisting of a catalytic core (domain I and II) and a helical domain III [10] . In this study, recombinant MERS-CoV main protease (M pro ) was expressed and purified; various spectroscopic techniques were used for characterizing structural and thermodynamic properties of MERS-CoV M pro at pH 1.0-7.0. The folded proteins are stabilized by different forces; therefore, characterizing protein stability is a significant challenge. Several methods (temperature, pH, chaotrops, ionic strength etc.) are used to characterize the protein stability [11] . In this study, we have tested the role of protonation in protein stability. In addition, insilico tools were used for comparative analysis of MERS-CoV M pro , with its closest homologs (SARS-CoV M pro and SARS-CoV-2 M pro ) at the sequence, structural and physicochemical level. J o u r n a l P r e -p r o o f Thermomixer was from Eppendorf, and Circular dichroism spectroscopy (CD) was from Jasco. E.coli BL21(DE3)pLysS harboring codon-optimized pET 3a M pro (Genscript) was used to express MERS-CoV M pro . The expression and extraction of MERS-CoV M pro from E.coli were performed as described in [10] . Briefly, recombinant MERS-CoV M pro was purified using Ni-NTA pre-packed column (1 ml, GE Healthcare). Initially, cleared crude lysate was passed through the column pre-equilibrated with 20 mM Tris pH 8.0, 500 mM NaCl, 20% glycerol, and 30 mM imidazole and washed with 10 column volumes equilibration buffer. Next, bound protein was eluted with elution buffer (equilibration buffer containing 500 mM imidazole) at 1 ml min -1 flow rate. The protein-containing fractions were pooled and analyzed by SDS-PAGE. Pure fractions were collected and stored at -80 o C after adding 10% glycerol. Before analysis, purified MERS-CoV M pro was thawed at room temperature and The protein was then diluted to 150 µg ml -1 with different buffers (pH 1.0 to 7.0) and equilibrated overnight at room temperature. The following buffers (30 mM each) were used; KCl-HCl (pH 1.0); Glycine-HCl (pH 2.0 and 3.0); Acetate (pH 4.0 and 5.0) and phosphate buffer (pH 6.0 and 7.0). scanning the emission intensity between 300-400 nm with slit width at 2.5 nm. The thermal stability of MERS-CoV M pro at different pHs was measured at 280 nm with 10 nm slit width, and the emission spectra were recorded between 310-360 nm (slit width 10 nm) Circular dichroism measurements were carried out by Jasco J-1500 CD spectropolarimeter. The temperature of the solutions was maintained at 25°C. Far-UV CD spectra were recorded in the wavelength range (200-250 nm) using a quartz cuvette of 1 mm path-length filled with 0.4 ml of MERS-CoV M pro (75 µg ml -1 ) at different pH (1.0-7.0). The amino acid sequences of different coronavirus (Covid-19 M pro , GenBank: QRX35868.1; SARS M pro , GenBank: AAR87511.1; MERS M pro , GenBank: QLD98008.1) mainproteases were retrieved from the UniProt database [12] . Sequence similarity searches were performed using Position-Specific Iterated BLAST (PSI-BLAST) [13] . MERS-CoV M pro was overexpressed in E. coli BL21 (DE3) pLysS and purified via affinity chromatography as described in Bo-Lin et al. protocol [10] . In addition, the purity of eluted fractions was analyzed on SDS-PAGE. As shown in Fig. 1 , MERS-CoV M pro was expressed at a right size of 35.5 kDa. Changes Rayleigh light scattering was performed to investigate pH-dependent MERS-CoV M pro aggregation. Light scattering of samples was monitored at 350 nm after excitations of the same wavelength. As shown in Fig. 4 , no significant increase in scattering intensity at 350 nm was observed across the entire pH range. Thus, MERS-CoV M pro does not form aggregates at any pH. The thermal stability of MERS-CoV M pro at pH 1.0-7.0 was monitored at 280 nm. The temperature was increased gradually from 20 to 90°C at a constant rate of 1 °C min -1 , and the ratio of 350 nm/330 nm was plotted with respect to temperature (Fig. 5) . Table 1 . Far UV-CD measurements studied changes in the secondary structure of MERS-CoV Interestingly at pH 1.0, MERS-CoV M pro regained native-like secondary structure. According to BLAST, MERS-CoV M pro was conserved, with 100% identity among all MERS-CoV variants with the query cover of 100%, and 0 E-value (Supplementary fig. S1 ). Table 3 . Analysis of physicochemical properties revealed that the three main proteases, polypeptides are 306 amino acids long with a molecular weight of around 33.0 KDa. According to the theoretical pI, all M pro s were negatively charged. The estimated half-life was more than 10 hours for all M pro . The instability index value for all M pro s was found as stable. The aliphatic index showed that SARS-CoV-2 M pro more thermally stable than MERS-CoV M pro and SARS-CoV M pro and a GRAVY was found negative in both Covid-19 M pro and SARS-CoV M pro . J o u r n a l P r e -p r o o f Preparing a purified and stable protein sample is a necessary procedure and is required before placing significant efforts into structural and biophysical studies. His-tagged recombinant MERS-CoV M pro was successfully expressed in E.coli and purified by using affinity chromatography in this study. Biomass was prepared by following an optimized protocol [10] . His-tagged MERS-CoV M pro was purified using Ni-NTA resin and detected by SDS-PAGE, and the position of the band was consistent with the calculated molecular weight of MERS-CoV M pro (Fig 1) . This indicated that the target protein was expressed, and the His-tag expression system was suitable for purifying MERS-CoV M pro . Biophysical methods have been widely used to understand the structural properties, folding and protein stability [17, 18] . This study employed a combination of techniques, including intrinsic and extrinsic fluorescence, Rayleigh scattering measurements, thermal shift assay, and far-UV CD. With these spectroscopic measurements, a pH range was applied to induce structural changes in the MERS-CoV M pro . Intrinsic tryptophan fluorescence provides information about the local microenvironment of the tryptophan residue, which response very sensitively to any change of the protein tertiary structure [19] . The maximum fluorescent intensity of MERS-CoV M pro at acidic pH 5.0 was observed at 330 nm (Fig 2) , suggesting that the tryptophan residues moved towards a more hydrophobic environment. At pH 2.0 the maximum fluorescence wavelength at 336 nm was observed, suggesting that the tertiary structure of MERS-CoV M pro was partially unfolded at this pH. The wavelength maximum of MERS-CoV M pro was red-shifted when pH lowered from neutral pH to pH 2.0. The red-shift of the maximum fluorescence peak resulted from changes in the tryptophan residues J o u r n a l P r e -p r o o f microenvironment from a non-polar to the solvent-exposed environment during the unfolding of the protein [20] . An extrinsic fluorescence dye (ANS) was used in this study. This extrinsic dye in an aqueous solution shows low fluorescence signals, but the fluorescence signals increase when exposed to a hydrophobic environment due to partially unfolded or aggregated proteins [21] . ANS emission spectra of MERS-CoV M pro (Fig 3) RLS intensity of a protein solution can be measured using a spectrofluorometer to detect the protein aggregation [30] . Our results showed that MERS-CoV M pro remained soluble at the entire pH range (Fig 4) . The thermal stability of MERS-CoV M pro was studied by thermal shift assay. MERS-CoV M pro at all pHs exhibited a single thermal transition (Fig 5) . Exceptionally at pH 2.0, no transitions were observed, and this result supports our finding that this protein obtained a partially unfolded state at pH 2.0. In a recent study, Covid-19 M pro also undergoes a single thermal transition with similar folding behavior of MERS-CoV M pro [25] . THE far-UV CD spectroscopy method is routinely used to rapidly determine the secondary structure of proteins and monitor dynamic changes of protein structure due to the straightforward J o u r n a l P r e -p r o o f sample preparation and fast acquisition time of this method [26] . In this study, the far-UV CD spectrum of MERS-CoV M pro at pH 7.0 (Fig 6) displayed two negative peaks at ~208 nm and ~222 nm, indicating alpha-helix dominant structure; the secondary structure was nearly intact up to pH 5.0. Below it, gain another minimum at 218 nm, which indicated a gain of beta-sheet-like structure in the MERS-CoV M pro at pH 2.0 and 3.0. Similar results were observed in previous studies of SARS-CoV and SARS-CoV-2 main proteases when calculated secondary structure contents [25] . In this study, we have also evaluated the similarity between the three main proteases of [27] . A recent study demonstrated that the superimposition of SARS-CoV and Covid-19 main proteases exhibit a high degree of active site conservation [9] . Analysis of physicochemical parameters was computed for MERS-CoV M pro , SARS-CoV M pro , and Covid-19 (Table 3) Protease inhibitors would be worth following as they may provide specific drugs for upcoming coronavirus outbreaks. 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