Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy
Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy
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DOI：
10.1002/cphc.200800210
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发表时间：
2008-09-15
期刊：
影响因子：
2.9
通讯作者：
Ando, Toshio
中科院分区：
文献类型：
--
作者：
Miyagi, Atsushi;Tsunaka, Yasuo;Ando, Toshio
蛋白质的内含子无序（ID）区域被认为参与生物学过程，如转录、翻译和细胞信号转导。尽管ID区域具有重要的作用，但没有有效的方法直接观察这些薄而灵活的结构。在这里，我们使用高速原子力显微镜（AFM）观察异二聚体的FACT（促进染色质转录）蛋白，这是预测在每个亚基有大的ID区域。FACT在云母表面上的连续AFM图像，以每秒5-17帧的速率捕获，清楚地显示了从FACT主体突出并在位置上波动的两个不同的类锯齿状部分。使用FACT的缺失突变体，我们将这些尾段鉴定为从氨基酸序列预测的两个主要ID区域。它们的力学性能估计从AFM图像表明，他们有更宽松的结构比随机线圈。这些观察结果表明，这种最先进的显微镜方法可用于表征难以用其他实验技术可视化的非结构化蛋白质片段。
Intrinsically disordered (ID) regions of proteins are recognized to be involved in biological processes such as transcription, translation, and cellular signal transduction. Despite the important roles of ID regions, effective methods to observe these thin and flexible structures directly were not available. Herein, we use high-speed atomic force microscopy (AFM) to observe the heterodimeric FACT (facilitates chromatin transcription) protein, which is predicted to have large ID regions in each subunit. Successive AFM images of FACT on a mica surface, captured at rates of 5-17 frames per second, clearly reveal two distinct toil-like segments that protrude from the main body of FACT and fluctuate in position. Using deletion mutants of FACT we identify these tail segments as the two major ID regions predicted from the amino acid sequences. Their mechanical properties estimated from the AFM images suggest that they have more relaxed structures than random coils. These observations demonstrate that this state-of-the-art microscopy method can be used to characterize unstructured protein segments that are difficult to visualize with other experimental techniques.