Cell Signaling and Inflammation Biology Lab, ICMR-National Institute of Pathology, New Delhi 110029, India.
Institute of Molecular Medicine, Jamia Hamdard, Hamdard Nagar, New Delhi, India; Department of Life Science, School of Basic Sciences and Research, Sharda University, Knowledge Park III, Greater Noida, Uttar Pradesh 201310, India.
Int J Biol Macromol. 2023 Aug 1;245:125455. doi: 10.1016/j.ijbiomac.2023.125455. Epub 2023 Jun 17.
Through comparative analyses using BLASTp and BLASTn of the 25 target sequences, our research identified two unique post-transcriptional modifiers, Rv1509 and Rv2231A, which serve as distinctive and characteristic proteins of M.tb - the Signature Proteins. Here, we have characterized these two signature proteins associated with pathophysiology of M.tb which may prove to be therapeutically important targets. Dynamic Light Scattering and Analytical Gel Filtration Chromatography exhibited that Rv1509 exists as a monomer while Rv2231A as a dimer in solution. Secondary structures were determined using Circular Dichroism and further validated through Fourier Transform Infrared spectroscopy. Both the proteins are capable of withstanding a wide range of temperature and pH variations. Fluorescence spectroscopy based binding affinity experiments showed that Rv1509 binds to iron and may promote organism growth by chelating iron. In the case of Rv2231A, a high affinity for its substrate RNA was observed, which is facilitated in presence of Mg suggesting it might have RNAse activity, supporting the prediction through in-silico studies. This is the first study on biophysical characterization of these two therapeutically important proteins, Rv1509 and Rv2231A, providing important insights into their structure -function correlations which are crucial for development of new drugs/ early diagnostics tools targeting these proteins.
通过对 25 个目标序列的 BLASTp 和 BLASTn 比较分析，我们的研究确定了两个独特的转录后修饰因子 Rv1509 和 Rv2231A，它们是 M.tb 的特征蛋白——Signature Proteins。在这里，我们对与 M.tb 病理生理学相关的这两种特征蛋白进行了表征，它们可能是具有治疗意义的重要靶点。动态光散射和分析凝胶过滤色谱显示，Rv1509 在溶液中以单体形式存在，而 Rv2231A 则以二聚体形式存在。使用圆二色性进一步通过傅里叶变换红外光谱确定二级结构。这两种蛋白质都能够耐受广泛的温度和 pH 变化。基于荧光光谱的结合亲和力实验表明，Rv1509 结合铁，通过螯合铁可能促进生物体生长。在 Rv2231A 的情况下，观察到其对底物 RNA 的高亲和力，在存在 Mg 的情况下促进了这种亲和力，表明它可能具有 RNAse 活性，这通过计算机模拟研究得到了预测。这是对这两种具有治疗意义的蛋白质 Rv1509 和 Rv2231A 的生物物理特性进行的首次研究，为它们的结构-功能相关性提供了重要的见解，这对于开发针对这些蛋白质的新药/早期诊断工具至关重要。
