Laboratory of Cell Morphology, Institute of Cytology Russian Academy of Science, St. Petersburg, Russia.
Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology Russian Academy of Science, St. Petersburg, Russia.
Prion. 2020 Dec;14(1):67-75. doi: 10.1080/19336896.2020.1720487.
Fluorescent probes thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) are widely used to study amyloid fibrils that accumulate in the body of patients with serious diseases, such as Alzheimer's, Parkinson's, prion diseases, etc. However, the possible effect of these probes on amyloid fibrils is not well understood. In this work, we investigated the photophysical characteristics, structure, and morphology of mature amyloid fibrils formed from two model proteins, insulin and lysozyme, in the presence of ThT and ANS. It turned out that ANS affects the secondary structure of amyloids (shown for fibrils formed from insulin and lysozyme) and their fibers clusterization (valid for lysozyme fibrils), while ThT has no such effects. These results confirm the differences in the mechanisms of these dyes interaction with amyloid fibrils. Observed effect of ANS was explained by the electrostatic interactions between the dye molecule and cationic groups of amyloid-forming proteins (unlike hydrophobic binding of ThT) that induce amyloids conformational changes. This interaction leads to weakening repulsion between positive charges of amyloid fibrils and can promote their clusterization. It was shown that when fibrillogenesis conditions and, consequently, fibrils structure is changing, as well as during defragmentation of amyloids by ultrasonication, the influence of ANS to amyloids does not change, which indicates the universality of the detected effects. Based on the obtained results, it was concluded that ANS should be used cautiously for the study of amyloid fibrils, since this fluorescence probe have a direct effect on the object of study.
荧光探针硫黄素 T（ThT）和 1-苯胺-8-萘磺酸（ANS）被广泛用于研究在患有严重疾病（如阿尔茨海默氏症、帕金森氏症、朊病毒病等）的患者体内积累的淀粉样纤维。然而，这些探针对淀粉样纤维的可能影响尚不清楚。在这项工作中，我们研究了两种模型蛋白（胰岛素和溶菌酶）形成的成熟淀粉样纤维在 ThT 和 ANS 存在下的光物理特性、结构和形态。结果表明，ANS 影响淀粉样物的二级结构（如胰岛素和溶菌酶形成的纤维）及其纤维聚集（如溶菌酶纤维），而 ThT 则没有这种影响。这些结果证实了这些染料与淀粉样纤维相互作用的机制存在差异。观察到的 ANS 效应是由染料分子与淀粉样形成蛋白的阳离子基团之间的静电相互作用引起的（与 ThT 的疏水性结合不同），这种相互作用诱导淀粉样物构象变化。这种相互作用削弱了淀粉样纤维正电荷之间的排斥力，并可以促进其聚集。结果表明，当纤维形成条件发生变化，进而改变纤维结构，以及通过超声处理使淀粉样物碎片化时，ANS 对淀粉样物的影响不变，这表明检测到的效应具有普遍性。基于获得的结果，得出结论认为，由于这种荧光探针对研究对象有直接影响，因此在研究淀粉样纤维时应谨慎使用 ANS。
