Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM
Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM
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DOI：
10.1091/mbc.e18-10-0661
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发表时间：
2019-06-01
影响因子：
3.3
通讯作者：
Heilemann, Mike
中科院分区：
文献类型：
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作者：
Baldering, Tim N.;Dietz, Marina S.;Heilemann, Mike
膜蛋白如何寡聚决定了它们的功能。超分辨率显微镜可以报告蛋白质聚类和提取定量分子信息。在这里，我们评估闪烁动力学的四个光活化荧光蛋白定量单分子显微镜。我们通过闪烁直方图分析鉴定mEos3.2和mMaple3适合于分子定量。我们设计了mEos3.2的合成和遗传二聚体以及单体和二聚体膜蛋白的融合蛋白作为参考结构，我们证明了它们在体外和原位定量超分辨率成像中的多用途。我们进一步发现mEos3.2和mMaple3的眨眼行为被还原剂修饰，提供了根据实验需要调整眨眼参数的可能性。
How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.