Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence
Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence
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DOI：
10.1074/jbc.c300049200
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发表时间：
2003-05-09
影响因子：
4.8
通讯作者：
Goto, Y
中科院分区：
文献类型：
--
作者：
Ban, T;Hamada, D;Goto, Y
实时监测淀粉样纤维生长对阐明淀粉样纤维形成机制至关重要。巯基黄素T （ThT）是一种已知的与淀粉样蛋白原纤维结合后产生强烈荧光的试剂。在这里，我们表明，通过全内反射荧光显微镜（TIRFM）监测ThT荧光，可以可视化β 2-微球蛋白（β 2-m）的淀粉样蛋白原纤维，而无需共价荧光标记。TIRFM的优点之一是我们可以选择性地监测沿着玻片的原纤维，这样我们就可以得到原纤维的确切长度。该方法用于跟踪种子依赖性β 2-m原纤维延伸动力学。这种延伸是单向的，速率不同，说明淀粉样蛋白结构具有异质性。由于ThT结合对所有淀粉样原纤维都是共同的，因此本方法将对淀粉样原纤维的分析具有普遍适用性。我们用从人体内提取的C端对应的八肽和阿尔茨海默氏淀粉样蛋白β肽证实了这一点。
Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.