Analysis of Red-Fluorescent Proteins Provides Insight into Dark-State Conversion and Photodegradation
Analysis of Red-Fluorescent Proteins Provides Insight into Dark-State Conversion and Photodegradation
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DOI：
10.1016/j.bpj.2011.06.055
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发表时间：
2011-08-17
影响因子：
3.4
通讯作者：
Palmer, Amy E.
中科院分区：
文献类型：
--
作者：
Dean, Kevin M.;Lubbeck, Jennifer L.;Palmer, Amy E.
荧光蛋白（FP）是一种强大的工具，可以实时可视化细胞过程。对于特定实验，给定FP的实用性强烈依赖于其有效亮度和整体光稳定性。然而，FPs的亮度受到暗态转换（DSC）和不可逆光漂白的限制，这发生在不同的时间尺度上。在这里，我们提出了在体内合奏测定DSC和不可逆的光漂白下连续和脉冲照明。对密切相关的红色FP的分析表明，DSC和不可逆光漂白并不总是通过相同的机制途径联系在一起。DSC发生在第一激发单重态之外，并且其幅度主要取决于从暗态恢复的动力学。实验结果可以通过电子态的四态模型的动力学模拟来复制。这里提出的方法允许光驱动的动力学进行研究，在合奏水平超过六个数量级的时间（微秒到秒的时间尺度）。
Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).