通过荧光相关和单分子光谱研究单体红色荧光蛋白中的暗态。
Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy.
作者信息
Hendrix Jelle, Flors Cristina, Dedecker Peter, Hofkens Johan, Engelborghs Yves
机构信息
Laboratory of Biomolecular Dynamics, Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium.
出版信息
Biophys J. 2008 May 15;94(10):4103-13. doi: 10.1529/biophysj.107.123596. Epub 2008 Jan 30.
Monomeric red fluorescent proteins (mRFPs) have become indispensable tools for studying protein dynamics, interactions and functions in the cellular environment. Their emission spectrum can be well separated from other fluorescent proteins, and their monomeric structure preserves the natural function of fusion proteins. However, previous photophysical studies of some RFPs have shown the presence of light-induced dark states that can complicate the interpretation of cellular experiments. In this article, we extend these studies to mRFP1, mCherry, and mStrawberry by means of fluorescence correlation spectroscopy and prove that this light-driven intensity flickering also occurs in these proteins. Furthermore, we show that the flickering in these proteins is pH-dependent. Single molecule spectroscopy revealed reversible transitions from a bright to a dark state in several timescales, even up to seconds. Time-resolved fluorescence spectroscopy showed multiexponential decays, consistent with a "loose" conformation. We offer a structural basis for the fluorescence flickering using known crystal structures and point out that the environment of Glu-215 is critical for the pH dependence of the flickering in RFPs. We apply dual-color fluorescence correlation spectroscopy inside live cells to prove that this flickering can seriously hamper cellular measurements if the timescales of the flickering and diffusion are not well separated.
单体红色荧光蛋白(mRFPs)已成为研究细胞环境中蛋白质动力学、相互作用和功能不可或缺的工具。它们的发射光谱能与其他荧光蛋白很好地分离,且其单体结构保留了融合蛋白的天然功能。然而,先前对一些红色荧光蛋白的光物理研究表明存在光诱导暗态,这可能会使细胞实验的解释变得复杂。在本文中,我们通过荧光相关光谱法将这些研究扩展到mRFP1、mCherry和mStrawberry,并证明这种光驱动的强度闪烁也发生在这些蛋白中。此外,我们表明这些蛋白中的闪烁是pH依赖性的。单分子光谱揭示了在几个时间尺度上从亮态到暗态的可逆转变,甚至长达数秒。时间分辨荧光光谱显示多指数衰减,与“松散”构象一致。我们利用已知的晶体结构为荧光闪烁提供了一个结构基础,并指出Glu-215的环境对于红色荧光蛋白中闪烁的pH依赖性至关重要。我们在活细胞内应用双色荧光相关光谱法来证明,如果闪烁和扩散的时间尺度没有很好地分离,这种闪烁会严重妨碍细胞测量。