Structural Changes in Tubulin Sheets Caused by Immobilization on Solid Supports.
Structural Changes in Tubulin Sheets Caused by Immobilization on Solid Supports.
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固定在固体支持物上引起的微管蛋白片的结构变化。
DOI：
10.1021/acsomega.8b02475
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发表时间：
2018
期刊：
影响因子：
4.1
通讯作者：
Sheffield,EvanC
中科院分区：
文献类型：
--
作者：
Donhauser,ZacharyJ;Appadoo,Visham;Kliman,ElysaJ;Jobs,WilliamB;Sheffield,EvanC
在锌的存在下，蛋白质微管蛋白组装成二维片，这是一个有用的模型系统的研究微管蛋白和微管结构。微管蛋白片层是原子力显微镜研究的理想蛋白质结构，因为它们含有二维晶体蛋白质晶格，并保留了微管蛋白和微管的许多结构特征。然而，高分辨率成像需要非微扰固定到适当的成像基底上。在这份报告中，几个基板通常用于扫描探针显微镜进行评估，其能力，以有效地抑制微管蛋白片：云母，黄金，高度有序的热解石墨，和碳涂层的电子显微镜网格。我们假设，这些基板所呈现的不同分子间相互作用将影响吸附的微管蛋白片的形态以及其他污染吸附物的量。微管蛋白片成功地在所有这些基板上成像，并报告了结构表征。碳涂层的电子显微镜网格，它保留了良好的结构特征的片材，并从未聚合的微管蛋白的吸附污染量最少，获得了最一致的结果。用原子力显微镜获得的微管蛋白片的图像也与使用类似程序生产的片的已发表的电子显微照片相比。这项工作证明了在研究二维蛋白质晶体时评估底物效应的重要性，并确定了用于固定微管蛋白片的合适底物。
In the presence of zinc, the protein tubulin assembles into two-dimensional sheets that are a useful model system for the study of both tubulin and microtubule structure. Tubulin sheets present an ideal protein structure for study with atomic force microscopy because they contain a two-dimensional crystalline protein lattice and retain many of the structural features of tubulin and microtubules. However, high-resolution imaging requires nonperturbative immobilization onto an appropriate imaging substrate. In this report, several substrates commonly used for scanning probe microscopy are evaluated for their ability to effectively immobilize tubulin sheets: mica, gold, highly ordered pyrolytic graphite, and carbon-coated electron microscopy grids. We hypothesize that the different intermolecular interactions presented by these substrates will affect the morphology of adsorbed tubulin sheets as well as the amount of other contaminating adsorbates. Tubulin sheets were successfully imaged on all of these substrates and structural characterization is reported. The most consistent results were obtained on carbon-coated electron microscopy grids, which preserved fine structural features of the sheets and had the least amount of contamination from the adsorption of unpolymerized tubulin. Images of tubulin sheets obtained with atomic force microscopy also compare favorably with published electron micrographs of sheets produced using similar procedures. This work demonstrates the importance of assessing substrate effects when studying two-dimensional protein crystals and identifies suitable substrates for immobilizing tubulin sheets.