Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology & LOEWE Center for Synthetic Microbiology (SYNMIKRO), Karl-von-Frisch-Str. 16, 35043, Marburg, Germany.
Institut für Assyriologie, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, 80539, München, Germany.
Angew Chem Int Ed Engl. 2017 Sep 11;56(38):11634-11639. doi: 10.1002/anie.201702870. Epub 2017 Jul 17.
Photoconversion of fluorescent proteins by blue and complementary near-infrared light, termed primed conversion (PC), is a mechanism recently discovered for Dendra2. We demonstrate that controlling the conformation of arginine at residue 66 by threonine at residue 69 of fluorescent proteins from Anthozoan families (Dendra2, mMaple, Eos, mKikGR, pcDronpa protein families) represents a general route to facilitate PC. Mutations of alanine 159 or serine 173, which are known to influence chromophore flexibility and allow for reversible photoswitching, prevent PC. In addition, we report enhanced photoconversion for pcDronpa variants with asparagine 116. We demonstrate live-cell single-molecule imaging with reduced phototoxicity using PC and record trajectories of RNA polymerase in Escherichia coli cells.
蓝光和近红外互补光的光致变色作用可将荧光蛋白转化为简体中文，称为引发转化（PC），这是 Dendra2 最近发现的一种机制。我们证明，通过在来自 Anthozoan 家族的荧光蛋白（Dendra2、mMaple、Eos、mKikGR、pcDronpa 蛋白家族）的第 66 位精氨酸上引入苏氨酸，可以控制第 69 位氨基酸的构象，这代表了促进 PC 的一般途径。已知影响生色团灵活性并允许可逆光开关的第 159 位丙氨酸或第 173 位丝氨酸的突变会阻止 PC 的发生。此外，我们报告了具有天冬酰胺 116 的 pcDronpa 变体的光致变色增强。我们演示了使用 PC 进行的活细胞单分子成像，其光毒性降低，并记录了大肠杆菌细胞中 RNA 聚合酶的轨迹。
