Savita Brajesh Kumar, Dalal Vikram, Choudhary Shweta, Gupta Deena Nath, Das Neeladrisingha, Tomar Shailly, Kumar Pravindra, Roy Partha, Sharma Ashwani Kumar
Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, 247 667, India.
Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, 247 667, India.
Biochem Biophys Res Commun. 2021 Nov 26;580:28-34. doi: 10.1016/j.bbrc.2021.09.076. Epub 2021 Sep 29.
The native pumpkin 2S albumin, a multifunctional protein, possess a variety of potential biotechnologically exploitable properties. The present study reports the characterization of recombinant pumpkin 2S albumin (rP2SA) and unraveling of its potential DNA/RNA binding site. The purification and characterization of the rP2SA established that it retains the characteristic α-helical structure and exhibited comparable DNase, RNase, antifungal and anti-proliferative activities as native protein. In vitro studies revealed that rP2SA exhibits potent antiviral activity against chikungunya virus (CHIKV) at a non-toxic concentration with an IC of 114.5 μg/mL. In silico studies and site-directed mutagenesis were employed to unravel the potential DNA/RNA binding site. A strong positive charge distribution due to presence of many arginine residues in proximity of helix 5 was identified as a potential site. The two of the arginine residues, conserved in some 2S albumins, were selected for the mutation studies. The mutated forms of recombinant protein (R84A and R91A) showed a drastic reduction in DNase and RNase activities suggesting their presence at binding site and involvement in the nuclease activity. A metal binding site was also identified adjacent to DNA/RNA binding site. The present study demonstrated the structural and functional integrity of the rP2SA and reports potential antiviral activity against CHIKV. Further, potential DNA/RNA binding site was unraveled through mutation studies and bioinformatics analysis.
天然南瓜2S白蛋白是一种多功能蛋白质，具有多种潜在的可用于生物技术开发的特性。本研究报告了重组南瓜2S白蛋白（rP2SA）的特性表征及其潜在DNA/RNA结合位点的解析。rP2SA的纯化和特性表征表明，它保留了特征性的α-螺旋结构，并表现出与天然蛋白质相当的脱氧核糖核酸酶、核糖核酸酶、抗真菌和抗增殖活性。体外研究表明，rP2SA在无毒浓度下对基孔肯雅病毒（CHIKV）表现出强大的抗病毒活性，半数抑制浓度为114.5μg/mL。采用计算机模拟研究和定点诱变来解析潜在的DNA/RNA结合位点。由于在螺旋5附近存在许多精氨酸残基而导致的强正电荷分布被确定为一个潜在位点。在一些2S白蛋白中保守的两个精氨酸残基被选用于突变研究。重组蛋白的突变形式（R84A和R91A）显示脱氧核糖核酸酶和核糖核酸酶活性急剧降低，表明它们存在于结合位点并参与核酸酶活性。还在DNA/RNA结合位点附近鉴定出一个金属结合位点。本研究证明了rP2SA的结构和功能完整性，并报告了其对CHIKV的潜在抗病毒活性。此外，通过突变研究和生物信息学分析解析了潜在的DNA/RNA结合位点。
