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A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Expression, purification, and biophysical characterization of recombinant MERS-CoV main (Mpro) protease
Ghada Obeid Almutairi1, Ajamaluddin Malik1, Mona Alonazi1
1Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia.
Abstract:
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.
Insights
This study characterizes the structural stability of MERS-CoV main protease (Mpro) under varying pH and temperature. MERS-CoV Mpro unfolds at acidic pH, forming a molten globule-like state, crucial for antiviral drug development.
Area of Science:
- Biochemistry
- Structural Biology
- Virology
Background:
- Middle East Respiratory Syndrome Coronavirus (MERS-CoV) main protease (Mpro) is vital for viral maturation and a key target for antiviral therapies.
- Understanding MERS-CoV Mpro conformation under different conditions is critical for drug design, but remains poorly characterized.
- Recombinant MERS-CoV Mpro was produced to investigate its structural properties.
Purpose of the Study:
- To characterize the structural stability of MERS-CoV Mpro at different pH and temperature conditions.
- To elucidate the conformational changes of MERS-CoV Mpro using spectroscopic methods.
- To provide thermodynamic and structural insights into MERS-CoV Mpro.
Main Methods:
- Recombinant production of MERS-CoV Mpro in E. coli.
- Intrinsic and extrinsic fluorescence spectroscopy to assess tertiary and secondary structure changes.
- Differential scanning fluorometry (DSF) to determine thermal stability and transitions.
- In silico analysis to compare MERS-CoV Mpro with other coronavirus proteases.
Main Results:
- Fluorescence measurements indicated unfolding of the tertiary structure of MERS-CoV Mpro in polar environments.
- At low pH, MERS-CoV Mpro exhibited enhanced secondary structure due to charge-charge repulsion.
- Differential scanning fluorometry revealed a molten globule-like state at pH 2.0, with no thermal transition observed.
- In silico analysis showed significant sequence similarity between MERS-CoV Mpro and SARS-CoV Mpro.
Conclusions:
- MERS-CoV Mpro undergoes significant structural changes, including tertiary unfolding and secondary structure gain at low pH.
- The protease forms a distinct molten globule-like state at highly acidic pH (2.0).
- These findings provide fundamental thermodynamic and structural data essential for developing targeted antiviral drugs against MERS-CoV.
