Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Department of Molecular Immunology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
ACS Chem Biol. 2021 May 21;16(5):891-904. doi: 10.1021/acschembio.1c00100. Epub 2021 Apr 29.
Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.
荧光蛋白标记系统在原核生物中的发展不如真核细胞系统成熟。在这里，我们通过引入基于转录因子的标签 LmrR 和 RamR，扩展了细菌中非共价荧光蛋白标签的概念，用于在需氧和厌氧条件下进行探针结合和荧光读出。我们开发了两种化学生物学蛋白标签，赋予可逆结合的有机荧光团荧光性和更长的荧光寿命，因此称为具有探针交换的化学生物学标签 (CTPEs)。我们对与两种不同 CTPE 可逆相互作用的 30 种荧光团进行了广泛的表征，并得出结论，芳香族平面结构与这些标签的疏水性口袋具有高度特异性结合。有机荧光团与 CTPE 的可逆结合以及有机荧光团的优越光物理性质，使得活细菌细胞的荧光显微镜能够长期观察。我们的蛋白标签为研究（亚）细胞蛋白定位和动态、蛋白-蛋白相互作用以及延长活细胞显微镜观察提供了一种通用工具，即使在无氧条件下也是如此。
