Expression, purification, and biophysical characterization of recombinant MERS-CoV main (Mpro) protease.

MERS-CoV main protease (Mpro) 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 Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro 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 Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro 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 Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro.

Introduction

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 21st 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 papain-like cysteine protease (PLpro) and main protease (Mpro, also called 3C-like cysteine protease, 3CLpro). Polyproteins pp1a and pp1b are cleaved by Mpro, which cleaves 11 sites, and PLpro, which cleaves at three sites, to release sixteen mature nonstructural proteins involved in coronaviruses transcription and replication [8], [9]. Both Mpro and PLpro, play an essential role in viral replication and maturation, making them attractive targets for discovering antiviral drugs [7], [9].

MERS-CoV Mpro (3CLpro) 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 (Mpro) [9]. MERS-CoV Mpro 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 (Mpro) was expressed and purified; various spectroscopic techniques were used for characterizing structural and thermodynamic properties of MERS-CoV Mpro 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 Mpro, with its closest homologs (SARS-CoV Mpro and SARS-CoV-2 Mpro) at the sequence, structural and physicochemical level.

Methods

Chemicals and instruments

E. coli BL21(DE3)pLysS was used to express recombinant protein from Invitrogen; glycerol and NaCl came from Scharlau; Chicken egg white lysozyme was from Fluka; ANS, ampicillin, and benzonase were from Sigma and IPTG was purchased from Biobasic. NuPAGE 4–12% bis- tris-gel from Life technologies; pre-packed Ni-NTA column was from GE Healthcare Life Sciences. Cary 60 UV–Vis spectrometer and Cary Eclipse spectrofluorometer were from Agilent technologies, Innova 44R Shaking incubator was from New Brunswick, Thermomixer was from Eppendorf, and Circular dichroism spectroscopy (CD) was from Jasco.

Expression and purification of MERS-CoV Mpro in E.coli BL 21(DE3)pLysS

E.coli BL21(DE3)pLysS harboring codon-optimized pET 3a Mpro (Genscript) was used to express MERS-CoV Mpro. The expression and extraction of MERS-CoV Mpro from E.coli were performed as described in [10]. Briefly, recombinant MERS-CoV Mpro 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 °C after adding 10% glycerol.

MERS-CoV Mpro quantification

Before analysis, purified MERS-CoV Mpro was thawed at room temperature and concentrated by using centricon tube at 4000 rpm for 40 min at 4 °C with three times buffer exchange (10 mM Tris pH 7.0, 50 mM NaCl). The absorbance of MERS-CoV Mpro was estimated at 280 nm, and the protein concentration was determined using an extinction coefficient of 43,890 M−1 cm−1.

Exposure of MERS-CoV Mpro to different pHs

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).

Fluorescence spectroscopy

Fluorescence spectra of MERS-CoV Mpro (1 μM) at different pH (1.0–7.0) were measured in a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) in a 1-cm path-length cuvette. The MERS-CoV Mpro samples were excited at 280 nm, and the fluorescence spectra were recorded in the range of 300–400 nm. Both excitation and emission slit widths were fixed at 10 nm. The changes in the surface hydrophobicity of MERS-CoV Mpro (1 μM) at different pHs were measured by adding 20 μM 8-Anilinonaphthalene-1-sulfonic acid (ANS). The samples were incubated at room temperature in the dark for 30 min before recording the fluorescence spectra. MERS-CoV Mpro samples at different pHs were excited at 385 nm (slit width set at 10 nm) and emission spectra were measured between 400 and 650 nm (slit width set at 10 nm). For aggregation studies of MERS-CoV Mpro at different pHs, Rayleigh scattering (RLS) measurement was performed at 25 °C by exciting samples at 350 nm (slit width 1.5 nm) and scanning the emission intensity between 300 and 400 nm with slit width at 2.5 nm.

Thermal shift assay

The thermal stability of MERS-CoV Mpro at different pHs was measured at 280 nm with 10 nm slit width, and the emission spectra were recorded between 310 and 360 nm (slit width 10 nm) in a Cary Eclipse Fluorescence Spectrophotometer. The temperature gradient used was 20–90 °C at a rate of 1 °C min−1. The T m values of MERS-CoV Mpro samples were obtained from the midpoint of the melting curve transition.

Circular dichroism (CD) measurements

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 Mpro (75 μg ml−1) at different pH (1.0–7.0).

Insilico studies of Mpro

The amino acid sequences of different coronavirus (Covid-19 Mpro, GenBank: QRX35868.1; SARS Mpro, GenBank: AAR87511.1; MERS Mpro, GenBank: QLD98008.1) main-proteases were retrieved from the UniProt database [12]. Sequence similarity searches were performed using Position-Specific Iterated BLAST (PSI-BLAST) [13]. Closely related sequences of MERS-CoV Mpro, SARS-CoV Mpro, and SARS-CoV-2 Mpro were downloaded from the BLAST database. Multiple sequence alignment and visualization were performed using Jalview program version 2.11.1.3 [14]. The 3D structure and critical catalytic residues of MERS-CoV Mpro (PDB: 5C3N), SARS-CoV Mpro (PDB: 2BX4), and SARS-CoV-2 Mpro (PDB: 6Y2E) were analyzed using PyMOL. The physical and chemical characteristics (molecular weight, theoretical pI, amino acid composition, half-life, instability index, aliphatic index, and grand average of hydropathicity index (GRAVY)) of MERS-CoV Mpro, SARS-CoV Mpro, and SARS-CoV-2 Mpro were investigated using the ProtParam tool [15].

Results

Expression and purification of recombinant MERS-CoV Mpro in E. coli

MERS-CoV Mpro 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 Mpro was expressed at a right size of 35.5 kDa.

Fig. 1

Purification of His-tagged MERS-CoV Mpro. Lane 1, marker; lane 2, total cell lysate; lane 3, flow-through; lane 4, wash; lane 5, fraction 1.

Intrinsic fluorescence

Changes in the tertiary structure of MERS-CoV Mpro were monitored by analyzing changes in the intrinsic fluorescence emission. Intrinsic fluorescence usually occurs due to the fluorescent emission of tyrosine and tryptophan when excited at 280 nm [16]. Intrinsic fluorescence spectra of MERS-CoV Mpro at different pHs are shown in (Fig. 2A). The fluorescence spectra of the MERS-CoV Mpro at pH 7 showed maximum fluorescence intensity at 330 nm, which is the characteristic feature of folded proteins. However, MERS-CoV Mpro showed the highest fluorescent intensity (I max) at pH 5.0 compared to at pH 7.0 at the same wavelength, i.e., 330 nm, indicating aromatic residues in the MERS-CoV Mpro moved towards a more hydrophobic environment. At pH 2.0, wavelength maxima (λ max) was red-shifted almost 6 nm from 330 to 336 nm, indicating that the tertiary structure of MERS-CoV Mpro destabilized due to unfolding (Fig. 2B). Initially, the microenvironment around aromatic residues was relatively unchanged between pH 7.0 to 4.0. However, its conformation was destabilized below pH 4.0. Further protonation below pH 2.0 leads to gain of the native-like structure, as the λ max was shifted back to native-like protein.

Fig. 2

Intrinsic fluorescence spectra of MERS-CoV Mpro. (A) Emission spectra of MERS-CoV Mpro at different pHs. An excitation wavelength of 280 nm was used and monitored emission in the range of 300–400 nm. (B) λmax plotted with respect to pH.

ANS binding assay

Extrinsic fluorescence was performed to identify the exposure of the hydrophobic patches on the MERS-CoV Mpro at different pHs. ANS is a hydrophobic sensitive dye that binds with proteins' hydrophobic regions. The ANS fluorescence intensity was found to increase when it was attached to the exposed hydrophobic patches. MERS-CoV Mpro at pH 7.0 showed lower ANS fluorescence but at pH 2.0 showed the highest fluorescent intensity (Imax), suggesting that MERS-CoV Mpro at pH 2.0 is in a partially unfolded state (Fig. 3A). The ANS fluorescence intensity was plotted against different pHs (Fig. 3B). From Fig. 3B, it was seen that the ANS fluorescence intensity was slightly increased in the range of pH 7.0 to 4.0 (Fig. 3B). Below pH 4.0, ANS intensity was increased quickly, indicating a large increase in the surface hydrophobicity and maximum fluorescence intensity was found at pH 2.0. Later on, the ANS fluorescence intensity at pH 1.0 was returned to the fluorescence intensity of native MERS-CoV Mpro, which designated that the MERS-CoV Mpro attained native-like folds.

Fig. 3

Extrinsic fluorescence spectra of MERS-CoV Mpro. (A) Binding of ANS with MERS-CoV Mpro at different pHs. Samples were excited at 385 nm, and the spectra were recorded from 400 to 650 nm. (B) Imax plotted with respect to pH.

Rayleigh light scattering (RLS) measurements

Rayleigh light scattering was performed to investigate pH-dependent MERS-CoV Mpro 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 Mpro does not form aggregates at any pH.

Fig. 4

Analysis of aggregate formation in MERS-CoV Mpro at pH 1.0–7.0.by RLS. MERS-CoV Mpro excited at 350 nm and emission spectra recorded between 300 and 400 nm.

The thermal stability of MERS-CoV Mpro 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 ). MERS-CoV Mpro at all pHs shows a single transition, except at pH 2.0 no changes were observed, and this result suggests that MERS-CoV Mpro at pH 2.0 lose tertiary structure and become partially unfolded. The thermal transition at pH 1.0 was very similar to the thermal transition of MERS-CoV Mpro at pH 7.0. The thermal shift assay also supports that the MERS-CoV Mpro at pH 2.0 is partially unfolded, and protein at pH 1.0 attained native-like stability. The thermal melting point (T ) of MERS-CoV Mpro at different pHs is shown in Table 1 .

Fig. 5

The thermal shift assay for MERS-CoV Mpro at pH 1.0–7.0. MERS-CoV Mpro samples were continuously heated from 20 to 90 °C at 1 °C min−1 and the spectra were collected at the range of 310–360 nm. The ratio of 350 nm/330 nm was plotted as a function of temperature.

Table 1

Thermal melting point (T) of MERS-CoV Mpro at pH 1.0, 4.0, 5.0, 6.0 and 7.0.

pH	Tm (°C)
1.0	61.9
2.0	N·D.
3.0	44.0
4.0	47.0
5.0	54.2
6.0	60.5
7.0	62.0

N.D: not determined.

Circular dichroism (CD)

Far UV-CD measurements studied changes in the secondary structure of MERS-CoV Mpro. The far-UV CD was measured in the range of 200–250 nm wavelength. The far-UV CD spectra of MERS-CoV Mpro at all pHs were plotted in Fig. 6 . From Fig. 6, it was clear that the far-UV CD spectrum of MERS-CoV Mpro at pH 7.0 was characterized by two negative peaks at 208 and 222 nm, which is a characteristic feature of the α-helix. The secondary structure was nearly intact up to pH 5.0. Below it, there was a gain of another minimum at 218 nm, which indicated a formation of beta-sheet-like structure in the MERS-CoV Mpro at pH 2.0 and 3.0. Interestingly at pH 1.0, MERS-CoV Mpro regained native-like secondary structure.

Fig. 6

Far UV CD studies of MERS-CoV Mpro. Far-UV CD spectra (200-250 nm) of 75 μg ml−1 MERS-CoV Mpro at pH 1.0 to 7.0.

Insilico studies of coronaviruses main protease

According to BLAST, MERS-CoV Mpro was conserved, with 100% identity among all MERS-CoV variants with the query cover of 100%, and 0 E-value (Supplementary fig. S1). Similar results were observed among all SARS-CoV Mpro variants (Supplementary fig. S2) and SARS-CoV-2 Mpro variants (Supplementary fig. S3), indicating that the main protease was fully developed conserved within coronavirus sub-classes. Next, the MERS-CoV Mpro protein sequence was compared with its closest homologs (SARS-CoV and Covid-19). The results revealed that the number of amino acid residues was identical beginning from Ser1 to Gln306. SARS-CoV-2 Mpro shares identity percentages of 96.08% with Covid-19 Mpro, and the differences were at twelve positions in the sequence alignment. The sequence identity between Covid-19 Mpro and MERS-CoV Mpro was 50.65% (Table 2 , Supplementary fig. S4). These results showed that the Covid-19 Mpro shared a higher sequence homology towards SARS-CoV Mpro than MERS-CoV Mpro. Finally, three different structural comparisons were performed (Supplementary fig. S5). The pairwise structural imposition of MERS-CoV Mpro with SARS-CoV Mpro (Supplementary fig. S5A) and with Covid-19 Mpro (Supplementary fig. S5B) indicates a lower degree of structural similarity and conservation of the catalytic dyads (Cys 148 instead of Cys 145) (Supplementary fig. S6), while SARS-CoV Mpro and Covid-19 Mpro exhibit nearly identical structures (Supplementary fig. S5C) with similar catalytic dyad residues Cys145 and His41 (Supplementary fig. S6). ProtParam tool was used to compute parameters to analyze physicochemical properties of MERS-CoV Mpro, SARS-CoV Mpro, and Covid-19 Mpro. The results were shown in 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 Mpros were negatively charged. The estimated half-life was more than 10 h for all Mpro. The instability index value for all Mpros was found as stable. The aliphatic index showed that SARS-CoV-2 Mpro more thermally stable than MERS-CoV Mpro and SARS-CoV Mpro and a GRAVY was found negative in both Covid-19 Mpro and SARS-CoV Mpro.

Table 2

Protein BLAST result using SARS-CoV-2 Mpro as a query sequence.

Subject sequence	Query coverage	E-value	Identity
SARS-CoV Mpro	100%	0.0	96.08%
MERS-CoV Mpro	100%	5e-115	50.65%

Table 3

Physicochemical parameter results computed by ExPASy ProtParam.

Protein name	Number of amino acids	Molecular weightKDa	Theoretical PI	Total number of negatively charged residues (Asp+Glu)	Total number of positively charged residues (Arg + Ly)	half-life (hour)(E. coli)	Instability index (II)	Aliphatic index	GRAVYa
MERS-CoV Mpro	306	33.3	5.86	21	16	>10	27.33	79.90	0.129
SARS-CoV Mpro	306	33.8	6.22	26	23	>10	29.67	81.83	−0.049
SARS-CoV-2 Mpro	306	33.7	5.95	26	22	>10	27.65	82.12	−0.019

GRAVY means the Grand average of hydropathicity.

Discussion

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 Mpro 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 Mpro 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 Mpro (Fig. 1). This indicated that the target protein was expressed, and the His-tag expression system was suitable for purifying MERS-CoV Mpro.

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 Mpro. 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 Mpro 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 Mpro was partially unfolded at this pH. The wavelength maximum of MERS-CoV Mpro 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 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 Mpro (Fig. 3) displayed the highest fluorescent intensity at pH 2.0, confirming that MERS-CoV Mpro at pH 2.0 is partially unfolded. It has been reported that ANS dye has a higher affinity to the partially unfolded state or molten globule (MG) state of the protein than that of the native and denatured states. Research has demonstrated the acidic pH-induced MG state in many proteins such as apo-α-lactalbumin, lipopolysaccharide-binding protein, and retinol-binding protein [22], [23], [24]. Further protonation below pH 2.0 leads to the gain of the native-like structure. The changes in intrinsic fluorescence (internal microenvironment) and extrinsic ANS fluorescence (Surface hydrophobicity) of MERS-CoV Mpro at different pHs were similar.

RLS intensity of a protein solution can be measured using a spectrofluorometer to detect the protein aggregation [30]. Our results showed that MERS-CoV Mpro remained soluble at the entire pH range (Fig. 4). The thermal stability of MERS-CoV Mpro was studied by thermal shift assay. MERS-CoV Mpro 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 Mpro also undergoes a single thermal transition with similar folding behavior of MERS-CoV Mpro [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 sample preparation and fast acquisition time of this method [26]. In this study, the far-UV CD spectrum of MERS-CoV Mpro 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 Mpro 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 MERS-CoV, SARS-CoV, and Covid-19 by using insilico tools to compare proteins based on sequence, structure, and physical, and chemical properties. This screening would be beneficial to drug screening researchers. Sequence alignment reported in (Supplementary fig. S1,2 and 3), shows a striking degree of conservation at an amino acid level among all Mpro variants. By aligning the multiple amino acid sequences of MERS-CoV Mpro, SARS-CoV Mpro, and Covid-19 (Supplementary fig. S4), we observed that these enzymes showed low conservancy MERS-CoV Mpro show only 50.65% sequence similarity with Covid-19 Mpro. In contrast, Covid-19 Mpro shows a higher sequence identity with the previous SARS-CoV Mpro, which is consistent with previous research results.

The 3D structures of MERS-CoV Mpro, SARS-CoV Mpro, and Covid-19 displayed the three main proteases containing a catalytic dyad consisting of a His residue and a Cys residue. MERS-CoV Mpro pairwise structural imposition to SARS-CoV Mpro (Supplementary fig. S5A) and to Covid-19 (Supplementary fig. S5B) indicates a comparatively lower degree of structural similarity and conservation of the catalytic dyads. The difference was in the Cys148 [10]. The structural imposition of Covid-19 Mpro and SARS-CoV Mpro, showed a difference in twelve amino acids and the obtained results showed that Covid-19 Mpro has a catalytic dyad (Cys145 and His41) consistent with SARS-CoV Mpro (Cys-145 and His-41), which exists precisely at a similar location [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 Mpro, SARS-CoV Mpro, and Covid-19 (Table 3). Length and size (molecular weight) are the most fundamental characteristic of protein sequences. No variation in protein length and molecular weight were observed in our study among the three Mpros. The isoelectric point and charge are also important parameters for solubility and interaction. It was observed that the theoretical pI was >7 for all Mpro, which indicates that all proteases are negatively charged and that it can be precipitated in acidic medium. Regarding the instability index value, all Mpro is classified as stable (instability index >40). Another measure for the stability of proteins is the aliphatic index. The aliphatic index is known as the volume of the protein occupied by the aliphatic side chain, and an increase in the aliphatic index value is reported to enhance the protein thermostability [28].

The aliphatic index of SARS-CoV-2 Mpro was 82.12, when compared to other Mpro inferred that SARS-CoV-2 Mpro was more stable under a wide range of temperature conditions.

It is also essential to determine the hydrophobic or hydrophilic character of the protein. In the case of SARS-CoV-2 Mpro and SARS-CoV Mpro, GRAVY scores were negative values indicating a low hydrophobic nature and good solubility. On the other hand, the MERS-CoV Mpro GRAVY score was a positive value showing greater hydrophobicity in nature, and the protein is sparingly soluble in water. This comparative analysis at the sequence, structural and physicochemical properties level could prove valuable for producing broad-spectrum main protease inhibitors. Protease inhibitors would be worth following as they may provide specific drugs for upcoming coronavirus outbreaks.

Conclusion

In conclusion, MERS-CoV Mpro at pH 2.0 exhibited a partially unfolded state. In addition, the thermal shift assay revealed that MERS-CoV Mpro unfolds via a single thermal transition at pH 7.0, and no transition was observed at pH 2.0. Moreover, Far-UV CD showed that MERS-CoV Mpro displayed a mixture of an α-helix and β-sheet structure at pH 7.0, and the secondary structure was modified at pH 2.0. Interestingly MERS-CoV Mpro regained all its secondary structure at pH 1.0. Similar CD spectrum and thermal stability results were also observed in Covid-19 Mpro. Finally, the insilico screening showed that Mpro is highly conserved between SARS-CoV and SARS-CoV-2, much more so than MERS-CoV Mpro.

CRediT authorship contribution statement

Ghada Obeid Almutairi: Data curation, Writing.

Ajamaluddin Malik: Conceptualization, Supervision, Writing, review & editing.

Mona Alonazi: Supervision, review & editing.

Javed Masood Khan: Methodology, Formal analysis, review & editing.

Abdullah S. Alhomida: Resources,

Mohd Shahnawaz Khan: Resources, review & editing.

Amal M. Alenad: Resources, review & editing.

Nojood Altwaijry: Funding acquisition, review & editing.

Nouf Omar Alafaleq: Resources, review & editing.