Malik Ajamaluddin, Alsenaidy Mohammad A
Department of Biochemistry, Protein Research Chair, College of Science, King Saud University, PO Box 2455, Riyadh, 11451, Saudi Arabia.
Vaccines and Biologics Research Unit, Department of Pharmaceutics, College of Pharmacy, King Saud University, PO Box 2457, Riyadh, 11451, Saudi Arabia.
3 Biotech. 2017 Jun;7(2):100. doi: 10.1007/s13205-017-0744-3. Epub 2017 May 30.
Within a decade, MERS-CoV emerged with nearly four times higher case fatality rate than an earlier outbreak of SARS-CoV and spread out in 27 countries in short span of time. As an emerging virus, combating it requires an in-depth understanding of its molecular machinery. Therefore, conformational characterization studies of coronavirus proteins are necessary to advance our knowledge of the matter for the development of antiviral therapies. In this study, MERS-CoV papain-like protease (PL) was recombinantly expressed and purified. Thermal folding pathway and thermodynamic properties were characterized using dynamic multimode spectroscopy (DMS) and thermal shift assay. DMS study showed that the PL undergoes a single thermal transition and follows a pathway of two-state folding with T and van't Hoff enthalpy values of 54.4 ± 0.1 °C and 317.1 ± 3.9 kJ/mol, respectively. An orthogonal technique based on intrinsic tryptophan fluorescence also showed that MERS-CoV PL undergoes a single thermal transition and unfolds via a pathway of two-state folding with a T value of 51.4 °C. Our findings provide significant understandings of the thermodynamic and structural properties of MERS-CoV PL.
在十年内，中东呼吸综合征冠状病毒（MERS-CoV）出现了，其病死率比早期严重急性呼吸综合征冠状病毒（SARS-CoV）疫情高出近四倍，并在短时间内蔓延到27个国家。作为一种新兴病毒，对抗它需要深入了解其分子机制。因此，冠状病毒蛋白的构象表征研究对于推进我们在抗病毒治疗开发方面的认识是必要的。在本研究中，中东呼吸综合征冠状病毒木瓜样蛋白酶（PL）被重组表达和纯化。使用动态多模式光谱（DMS）和热位移分析对热折叠途径和热力学性质进行了表征。DMS研究表明，PL经历单一的热转变，并遵循两态折叠途径，其熔解温度（Tm）和范特霍夫焓值分别为54.4±0.1°C和317.1±3.9kJ/mol。基于内在色氨酸荧光的正交技术也表明，中东呼吸综合征冠状病毒PL经历单一的热转变，并通过两态折叠途径展开，其熔解温度值为51.4°C。我们的研究结果为中东呼吸综合征冠状病毒PL的热力学和结构性质提供了重要的认识。
