Department of Microbiology, Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, 420008 Kazan, Russia.
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia.
Int J Mol Sci. 2022 Feb 7;23(3):1867. doi: 10.3390/ijms23031867.
Small cationic guanyl-preferring ribonucleases (RNases) produced by the species share a similar protein tertiary structure with a high degree of amino acid sequence conservation. However, they form dimers that differ in conformation and stability. Here, we have addressed the issues (1) whether the homologous RNases also have distinctions in catalytic activity towards different RNA substrates and interactions with the inhibitor protein barstar, and (2) whether these differences correlate with structural features of the proteins. Circular dichroism and dynamic light scattering assays revealed distinctions in the structures of homologous RNases. The activity levels of the RNases towards natural RNA substrates, as measured spectrometrically by acid-soluble hydrolysis products, were similar and decreased in the row high-polymeric RNA >>> transport RNA > double-stranded RNA. However, stopped flow kinetic studies on model RNA substrates containing the guanosine residue in a hairpin stem or a loop showed that the cleavage rates of these enzymes were different. Moreover, homologous RNases were inhibited by the barstar with diverse efficiency. Therefore, minor changes in structure elements of homologous proteins have a potential to significantly effect molecule stability and functional activities, such as catalysis or ligand binding.
小型阳离子鸟苷偏好核糖核酸酶（RNases）由 种产生，它们具有相似的蛋白质三级结构，具有高度的氨基酸序列保守性。然而，它们形成的二聚体在构象和稳定性上存在差异。在这里，我们解决了以下问题：（1）同源 RNases 对不同 RNA 底物的催化活性以及与抑制剂蛋白 barstar 的相互作用是否存在差异，以及（2）这些差异是否与蛋白质的结构特征相关。圆二色性和动态光散射分析表明，同源 RNases 的结构存在差异。通过酸溶性水解产物的分光光度法测量，RNases 对天然 RNA 底物的活性水平相似，并且随着聚合 RNA > 转运 RNA > 双链 RNA 的顺序降低。然而，在含有发夹茎或环中的鸟苷残基的模型 RNA 底物上进行的停流动力学研究表明，这些酶的切割速率不同。此外，同源 RNases 被 barstar 以不同的效率抑制。因此，同源蛋白结构元件的微小变化有可能显著影响分子稳定性和功能活性，如催化或配体结合。
