Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins
Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins
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DOI：
10.1073/pnas.97.1.151
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发表时间：
2000-01-04
影响因子：
11.1
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Schwille, P;Kummer, S;Webb, WW
在10（-8）M下，用荧光相关光谱法研究了黄移绿色荧光蛋白突变体T203 Y和T203 F（含S65 G/S72 A）在10（-6）-10（-3）-s时间范围内荧光发射的快速激发驱动波动。这种强度依赖性闪烁是显着的，在高pH值，速率常数独立的pH值和粘度与轻微的温度效应。平均闪烁率随激发强度线性增加至少三十年，但经历这些动力学的分子的平均暗分数在约6 × 10（2）至5 × 10（6）W/cm（2）范围内与照明强度无关。这些结果表明，光激发建立了两个分子状态之间的平衡，不同的光谱特性，耦合仅通过激发态作为网关。这种可逆的激发驱动跃迁具有约10（-3）的量子效率。外部质子化的动力学，可逆地淬灭荧光，也观察到在低pH值在10- 100亩的时间范围内。这两个亮-暗闪烁过程的独立性意味着这些绿色荧光蛋白突变体至少存在两个独立的暗态。时间分辨荧光测量揭示了一个单一的指数衰减的激发态人口与3.8 ns的寿命，500 nm的激发后，这是pH独立。我们的荧光相关光谱的结果进行了讨论，在最近的理论研究，调用异构化的发色团作为激发态弛豫的非辐射通道。
Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10(-6)-10(-3)-s time range, by using fluorescence correlation spectroscopy at 10(-8) M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over approximate to 6 x 10(2) to 5 x 10(6) W/cm(2). These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of approximate to 10(-3). Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-mu s time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.