Heikal A A, Hess S T, Baird G S, Tsien R Y, Webb W W
School of Applied and Engineering Physics, Cornell University, Clark Hall, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11996-2001. doi: 10.1073/pnas.97.22.11996.
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 approximately 3 x 10(-3). Unlike Citrine (pKa approximately 5.7), 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 approximately 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 ( approximately 100 GM at 990 nm for dsRed and approximately 50 GM at 970 nm for Citrine), advantageous for two-photon-fluorescence imaging in cells, are measured.
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观察窗口，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，通过荧光相关光谱和时间相关单光子计数对来自盘珊瑚（珊瑚）的红色荧光蛋白（dsRed）的光物理性质进行了表征，该蛋白具有所需的更长发射/吸收波长，还对一种经过改进的黄色荧光蛋白突变体（柠檬黄）（S65G/V68L/Q69M/S72A/T203Y）进行了显著比较。dsRed荧光以3.65±0.07纳秒的时间常数呈单指数衰减，表明存在一个独立于pH 4.4 - 9.0的单一发射态/物种，这与柠檬黄不同。然而，激光激发会在暗态和亮态之间以10³ - 10⁴赫兹的频率驱动可逆荧光闪烁，其恒定分配分数f(1)=0.42±0.06，量子产率约为3×10⁻³。与柠檬黄（pKa约为5.7）不同，dsRed中pH依赖的质子结合在pH 3.9 - 11时可忽略不计。dsRed的时间分辨各向异性显示出快速去极化（211±6皮秒）加上缓慢的旋转运动（53±8纳秒），而柠檬黄只有单一的旋转时间（16±2纳秒）。根据旋转和平移扩散计算出的分子尺寸表明，dsRed的流体动力学尺寸比单体预测值大3.8±0.4倍。这表明即使在约10⁻⁹摩尔浓度下，dsRed也呈寡聚体（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寡聚体内能量转移，初始各向异性意味着去极化角度为24±3度。测量了较大的双光子激发截面（dsRed在990纳米处约为100 GM，柠檬黄在970纳米处约为50 GM），这有利于细胞中的双光子荧光成像。
