Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331-4003, USA.
Int J Mol Sci. 2022 Jun 9;23(12):6459. doi: 10.3390/ijms23126459.
The advancement of super-resolution imaging (SRI) relies on fluorescent proteins with novel photochromic properties. Using light, the reversibly switchable fluorescent proteins (RSFPs) can be converted between bright and dark states for many photocycles and their emergence has inspired the invention of advanced SRI techniques. The general photoswitching mechanism involves the chromophore - isomerization and proton transfer for negative and positive RSFPs and hydration-dehydration for decoupled RSFPs. However, a detailed understanding of these processes on ultrafast timescales (femtosecond to millisecond) is lacking, which fundamentally hinders the further development of RSFPs. In this review, we summarize the current progress of utilizing various ultrafast electronic and vibrational spectroscopies, and time-resolved crystallography in investigating the on/off photoswitching pathways of RSFPs. We show that significant insights have been gained for some well-studied proteins, but the real-time "action" details regarding the bidirectional - isomerization, proton transfer, and intermediate states remain unclear for most systems, and many other relevant proteins have not been studied yet. We expect this review to lay the foundation and inspire more ultrafast studies on existing and future engineered RSFPs. The gained mechanistic insights will accelerate the rational development of RSFPs with enhanced two-way switching rate and efficiency, better photostability, higher brightness, and redder emission colors.
超分辨率成像（SRI）的发展依赖于具有新型光致变色特性的荧光蛋白。利用光，可将可逆切换的荧光蛋白（RSFP）在亮态和暗态之间多次转换，其出现激发了先进 SRI 技术的发明。一般的光致开关机制涉及发色团 - 互变异构和质子转移用于负和正 RSFPs，以及去耦 RSFPs 的水合 - 去水合。然而，对这些超快时间尺度（飞秒到毫秒）下的过程缺乏详细的了解，这从根本上阻碍了 RSFPs 的进一步发展。在这篇综述中，我们总结了利用各种超快电子和振动光谱学以及时间分辨晶体学研究 RSFPs 开/关光致开关途径的最新进展。我们表明，对于一些研究较好的蛋白质已经获得了重要的见解，但对于大多数系统，关于双向互变异构、质子转移和中间态的实时“动作”细节仍然不清楚，并且许多其他相关蛋白质尚未得到研究。我们希望这篇综述能够为现有的和未来的工程化 RSFPs 的更多超快研究奠定基础并激发灵感。所获得的机制见解将加速具有增强的双向切换速率和效率、更好的光稳定性、更高的亮度和更红的发射颜色的 RSFPs 的合理发展。
