Imaging proteins sensitive to direct fusions using transient peptide-peptide interactions
Imaging proteins sensitive to direct fusions using transient peptide-peptide interactions
复制标题
使用瞬时肽-肽相互作用对直接融合敏感的蛋白质进行成像
DOI：
10.1101/2023.07.01.547312
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Gidden Z
中科院分区：
文献类型：
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作者：
Gidden Z
荧光显微镜使活细胞中的蛋白质特异性可视化，并在我们理解蛋白质亚细胞位置和功能方面发挥了重要作用。然而，当使用与荧光蛋白的直接融合进行标记时，一些蛋白质显示出改变的定位或功能，使得它们难以在活细胞中进行研究。此外，荧光显微镜的分辨率仅限于200 nm，这比大多数蛋白质的大小大2个数量级。为了规避这些挑战，我们之前开发了LIVE-PAINT，这是一种活细胞超分辨率方法，它利用短相互作用肽将荧光蛋白瞬时结合到感兴趣的蛋白质上。在这里，我们成功地使用LIVE-PAINT图像酵母膜蛋白，不容忍直接融合的荧光蛋白，通过使用短至5个残基的肽标签。我们还表明，它是可能的，同时解决多个蛋白质在纳米级使用正交肽相互作用对。
Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.