Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad , Hyderabad 500 046, India.
J Phys Chem B. 2018 Jan 25;122(3):1132-1144. doi: 10.1021/acs.jpcb.7b10561. Epub 2018 Jan 9.
When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two-state transitions during chemical denaturation. The extent of stability exerted by Glu is higher for RNase A at higher temperature, whereas it provides more stability for α-LA at lower temperature. Thus, the experiments indicate that Glu induces a thermal equilibrium intermediate and increases the thermodynamic stability of proteins irrespective of their surface charges. The extent of stability varies between the proteins in a temperature-dependent manner.
当生物体受到应激条件时，它们的一种适应反应是积累称为渗透物的小分子有机物质。这些渗透物会影响包括蛋白质在内的生物大分子的结构和稳定性。本研究检查了带负电荷的氨基酸渗透物谷氨酸（Glu）对两种模型蛋白，核糖核酸酶 A（RNase A）和α-乳白蛋白（α-LA）的影响，这两种蛋白在 pH 7 时分别带有正电荷和负电荷。这些蛋白质在热和化学诱导变性过程中都遵循两态展开转变。添加 Glu 可以稳定蛋白质免受温度影响，并在展开过程中诱导早期平衡中间体。发现稳定性是焓驱动的，并且与 RNase A 相比，α-LA 的稳定自由能更高。在存在 Glu 的情况下，两种蛋白质的偏摩尔体积和压缩性降低表明蛋白质通过表面水合作用达到更紧凑的状态，从而提供更稳定的构象。这也得到分子动力学模拟研究的支持，该研究表明，添加 Glu 后，蛋白质周围的水密度增加。此外，较低浓度（约 0.5 M）的盐酸胍和盐可完全使中间体失稳。然而，脲可以破坏α-LA 形成的 Glu 诱导的中间产物，而对于带正电荷的 RNase A，它只略微失稳，并且可能与 Glu 具有电荷-电荷相互作用。这表明，除了水合作用外，库仑相互作用也可能对中间产物的稳定性做出贡献。在不同温度下，在存在和不存在 Glu 的情况下，用 Gdm 对 RNase A 和 α-LA 进行变性。这些结果还表明 Glu 对两种蛋白质均具有稳定作用；然而，在化学变性过程中，所有展开转变均遵循两态转变。在较高温度下，Glu 对 RNase A 的稳定性更高，而在较低温度下，Glu 对α-LA 的稳定性更高。因此，实验表明，Glu 诱导热平衡中间产物并增加蛋白质的热力学稳定性，而与它们的表面电荷无关。在温度依赖性的方式下，两种蛋白质的稳定性程度不同。
