Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsRed) and yellow (Citrine)
Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsRed) and yellow (Citrine)
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DOI：
10.1073/pnas.97.22.11996
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发表时间：
2000-10-24
影响因子：
11.1
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Heikal, AA;Hess, ST;Webb, WW
生物系统中固有荧光蛋白的基因表达为细胞功能提供了新的非侵入性窗口，但这些探针的优化依赖于对其分子光谱，动力学和结构的理解。在这里，红色荧光蛋白（dsRed）从discosoma（珊瑚），提供所需的较长的发射/吸收波长，和一个改进的黄色荧光蛋白突变体（Citrine）（S65 G/V68 L/Q69 M/S72 A/T203 Y）的显着比较，其特征在于通过使用荧光相关光谱和时间相关的单光子计数。dsRed荧光以3.65 +/- 0.07-ns的时间常数作为单指数衰减，表明与Citrine相反，与pH 4.4-9.0无关的单一发射状态/物质。然而，激光激发在暗态和亮态之间以10（3）-10（4）Hz驱动可逆荧光闪烁，具有恒定的分配分数f（1）= 0.42 +/- 0.06和约3 × 10（-3）的量子产率。与Citrine不同（pKa约为5.7），dsRed中pH依赖性质子结合可忽略不计（pH 3.9-11）。dsRed的时间分辨各向异性揭示了快速去极化（211 +/-6ps）加上缓慢旋转运动（53 +/- 8 ns），与Citrine的单个旋转时间（16 +/-2ns）形成对比。根据旋转和平移扩散计算的分子尺寸表明，dsRed在流体动力学上比单体预测的大3.8 +/- 0.4倍，这表明即使在接近10（-9）M时也存在低聚物（可能是四聚体）构型。快速去极化归因于移动的非平行发色团之间的低聚物内能量转移，初始各向异性意味着24 +/-3度的去极化角。测量了大的双光子激发横截面（对于dsRed在990 nm处约为100 GM，对于Citrine在970 nm处约为50 GM），这对于细胞中的双光子荧光成像是有利的。
Gene expression of intrinsically fluorescent proteins in biological systems offers new noninvasive windows into cellular function, but optimization of these probes relies on understanding their molecular spectroscopy, dynamics, and structure. Here, the photophysics of red fluorescent protein (dsRed) from discosoma (coral), providing desired longer emission/absorption wavelengths, and an improved yellow fluorescent protein mutant (Citrine) (S65G/V68L/Q69 M/S72A/ T203Y) for significant comparison, are characterized by using fluorescence correlation spectroscopy and time-correlated single-photon counting. dsRed fluorescence decays as a single exponential with a 3.65 +/- 0.07-ns time constant, indicating a single emitting state/species independent of pH 4.4-9.0, in contrast with Citrine. However, laser excitation drives reversible fluorescence flicker at 10(3)-10(4) Hz between dark and bright states with a constant partition fraction f(1) = 0.42 +/- 0.06 and quantum yield of approximate to3 x 10(-3). Unlike Citrine (pKa approximate to5.7), pH-dependent proton binding is negligible (pH 3.9-11) in dsRed. Time-resolved anisotropy of dsRed reveals rapid depolarization (211 +/- 6 ps) plus slow rotational motion (53 +/- 8 ns), in contrast with a single rotational time (16 +/- 2 ns) for Citrine. The molecular dimensions, calculated from rotational and translational diffusion, indicate that dsRed is hydrodynamically 3.8 +/- 0.4 times larger than predicted for a monomer, which suggests an oligomer (possibly a tetramer) configuration even at approximate to 10(-9) M. The fast depolarization is attributed to intraoligomer energy transfer between mobile nonparallel chromophores with the initial anisotropy implying a 24 +/- 3 degrees depolarization angle. Large two-photon excitation cross sections (approximate to 100 GM at 990 nm for dsRed and approximate to 50 GM at 970 nm for Citrine), advantageous for two-photon-fluorescence imaging in cells, are measured.