Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany.
Abberior Instruments GmbH, Hans-Adolf-Krebs-Weg 1, 37077, Göttingen, Germany.
Sci Rep. 2018 Feb 9;8(1):2724. doi: 10.1038/s41598-018-19947-1.
The reversibly switchable fluorescent proteins (RSFPs) commonly used for RESOLFT nanoscopy have been developed from fluorescent proteins of the GFP superfamily. These proteins are bright, but exhibit several drawbacks such as relatively large size, oxygen-dependence, sensitivity to low pH, and limited switching speed. Therefore, RSFPs from other origins with improved properties need to be explored. Here, we report the development of two RSFPs based on the LOV domain of the photoreceptor protein YtvA from Bacillus subtilis. LOV domains obtain their fluorescence by association with the abundant cellular cofactor flavin mononucleotide (FMN). Under illumination with blue and ultraviolet light, they undergo a photocycle, making these proteins inherently photoswitchable. Our first improved variant, rsLOV1, can be used for RESOLFT imaging, whereas rsLOV2 proved useful for STED nanoscopy of living cells with a resolution of down to 50 nm. In addition to their smaller size compared to GFP-related proteins (17 kDa instead of 27 kDa) and their usability at low pH, rsLOV1 and rsLOV2 exhibit faster switching kinetics, switching on and off 3 times faster than rsEGFP2, the fastest-switching RSFP reported to date. Therefore, LOV-domain-based RSFPs have potential for applications where the switching speed of GFP-based proteins is limiting.
可反复开关的荧光蛋白（RSFP）常用于 RESOLFT 纳米显微镜，它们源自 GFP 超家族的荧光蛋白。这些蛋白亮度高，但存在一些缺点，例如相对较大的尺寸、对氧气的依赖性、对低 pH 值的敏感性以及有限的开关速度。因此，需要探索具有改进性能的来自其他来源的 RSFPs。在这里，我们报告了两种基于枯草芽孢杆菌光受体蛋白 YtvA 的 LOV 结构域的 RSFPs 的开发。LOV 结构域通过与丰富的细胞辅因子黄素单核苷酸（FMN）结合获得其荧光。在蓝光和紫外光的照射下，它们会经历光循环，使这些蛋白天生具有可光开关性。我们的第一个改进变体 rsLOV1 可用于 RESOLFT 成像，而 rsLOV2 则可用于活细胞的 STED 纳米显微镜，分辨率可达 50nm。与 GFP 相关蛋白（27 kDa 相比，rsLOV1 和 rsLOV2 的尺寸更小（17 kDa），并且在低 pH 值下也可用，并且其开关动力学更快，开关速度比迄今为止报道的最快开关 RSFP rsEGFP2 快 3 倍。因此，基于 LOV 结构域的 RSFPs 在 GFP 蛋白开关速度有限的应用中具有潜力。
