School of Biotechnology Jawaharlal Nehru University, New Delhi 110067, India.
School of Biotechnology Jawaharlal Nehru University, New Delhi 110067, India.
Biophys Chem. 2020 Sep;264:106422. doi: 10.1016/j.bpc.2020.106422. Epub 2020 Jun 30.
Intrinsically disordered proteins (IDPs) comprise ~30-40% of the proteome, have key roles in cellular processes, and have been reported to be involved in stress regulation working in synergy with osmolytes. Osmolytes are known to accumulate against various stresses in living systems and are known to stabilize the native conformation of globular proteins. However, little is known of their effect on IDPs and their mechanism of action is unclear. We have investigated the effect of a series of polyol osmolytes on the conformation, aggregation and fibrillation properties of the IDPs α and β-synuclein, involved in Parkinson's disease, using fluorescence, CD, light scattering and TEM. We observe inhibition of fibril and aggregate formation with increasing concentration as well as the number of hydroxyl groups in polyols as observed by light scattering measurements which correlates well with the increase in viscosity of solution with increasing number of OH groups in them. However, ThT assay, while indicating suppression of fibril formation at various concentrations of polyols, shows enhanced fibrillation at some other concentrations which could be due to the heterogeneity of the species formed that are ThT insensitive. Fibril formation was, thus, probed by using Nile red fluorescence which showed sensitivity towards the species formed. ANS binding fluorescence also indicates a decrease in the hydrophobicity of the fibrils with increasing number of OH groups in polyols. Polyols do not have any effect on the fibrillation of β-syn but lead to enhanced amorphous aggregate formation in presence of Ethylene Glycol and Glycerol and a reduction in the presence of Sorbitol. The net free energy of transfer of the proteins from water to Sorbitol is large and positive while it is relatively negligible in the case of Glycerol suggestive of greater preferential exclusion effect of Sorbitol in comparison with Glycerol in the case of IDPs as well. The results overall show differential and complex effect of osmolytes towards the fibrillation/aggregation properties of the two IDPs and suggest that an appropriate balance between the concentration and type of polyol or osmolyte would be required for the survival of organisms rich in IDPs under various stress conditions.
无定形蛋白质（IDPs）约占蛋白质组的 30-40%，在细胞过程中具有关键作用，并已报道与渗透剂协同作用参与应激调节。渗透剂已知在活系统中积累以应对各种应激，并且已知稳定球状蛋白质的天然构象。然而，对于 IDPs 的影响知之甚少，其作用机制尚不清楚。我们使用荧光、CD、光散射和 TEM 研究了一系列多元醇渗透剂对参与帕金森病的 IDPα和β-突触核蛋白的构象、聚集和纤维化特性的影响。我们观察到随着浓度的增加以及多元醇中羟基数量的增加，纤维和聚集体的形成受到抑制，这与随着 OH 基团数量的增加溶液粘度的增加相一致。然而，ThT 测定法虽然在各种多元醇浓度下表明抑制纤维形成，但在其他一些浓度下显示出增强的纤维形成，这可能是由于形成的物种的异质性导致的，这些物种对 ThT 不敏感。因此，通过使用尼罗红荧光探测纤维形成，该荧光对形成的物种敏感。ANS 结合荧光也表明，随着多元醇中 OH 基团数量的增加，纤维的疏水性降低。多元醇对β-突触核蛋白的纤维化没有影响，但导致在存在乙二醇和甘油的情况下增强无定形聚集体形成，而在存在山梨糖醇的情况下减少。蛋白质从水到山梨糖醇的自由能转移的净自由能很大且为正，而在甘油的情况下则相对可以忽略不计，这表明与甘油相比，山梨糖醇在 IDPs 中具有更大的优先排斥效应。总的来说，这些结果表明渗透剂对两种 IDP 的纤维化/聚集特性具有不同的复杂影响，并表明在各种应激条件下，富含 IDP 的生物体需要在浓度和多元醇或渗透剂的类型之间取得适当的平衡，以确保其生存。
