Fluorescence photoconversion kinetics in novel green fluorescent protein pH sensors (pHluorins)
Fluorescence photoconversion kinetics in novel green fluorescent protein pH sensors (pHluorins)
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DOI：
10.1021/jp0362077
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发表时间：
2004-07-15
影响因子：
3.3
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Hess, ST;Heikal, AA;Webb, WW
基因编码的pH敏感性绿色荧光蛋白（GFP）为细胞生物学和神经科学应用提供了对定位、光谱特性和环境敏感性的显著控制。生物应用的定量分析和新的GFP特性的充分利用依赖于对它们的分子生物物理学的基本理解。在这方面的贡献，接地和激发态荧光特性的pHluorins，两个pH敏感的GFP探针突触前活性，并与蓝宝石，中性苯酚GFP突变体进行比较。分子动力学已经使用荧光相关光谱（FCS）和时间相关单光子计数（TCSPC）表征为pH、激发波长（包括405 nm）、检测波长和照明强度的函数。作为细胞pH指示剂，与蓝宝石（H9; pK（a）= 5.7 +/-0.1）和许多其他GFP相比，磷酸钙（EcGFP）特别适合于生理pH范围（pK（a）= 7.2 +/-0.2）。然而，EcGFP和H9都表现出复杂的，强度和波长依赖性的明亮和黑暗的EcGFP状态之间的分区，这可能使它们在定量细胞内测量中的使用复杂化。此外，质子化和去质子化速率常数的EcGFP生色团可能会限制解决的能力，发生在时间尺度上的pH跳跃快于类似to0.5毫秒。一个建议的三态转换的动力学模型定量描述了通过可逆的内部和外部质子化的状态间转换。EcGFP衰变的激发中性态荧光寿命显着更长（即，更大的荧光量子产率），这可以作为一个新的光谱窗口，在生物学研究与多个标签。EcGFP中性荧光寿命的pH独立性意味着在酸性环境中观察到的荧光猝灭的基态相互转换机制。
Genetically encoded pH-sensitive green fluorescent proteins (GFPs) offer significant control over localization, spectral properties, and environmental sensitivity for cell biology and neuroscience applications. Quantitative analysis of biological applications and full exploitation of novel GFP properties rely on a fundamental understanding of their molecular photophysics. In this contribution, the ground- and excited-state fluorescence properties of Ecliptic and Ratiometric pHluorins, two pH-sensitive GFP probes of presynaptic activity, are presented and compared with Sapphire, a neutral phenol GFP mutant. Molecular dynamics have been characterized using fluorescence correlation spectroscopy (FCS) and time-correlated single-photon counting (TCSPC) as a function of pH, excitation wavelength (including 405 nm), detection wavelength, and illumination intensity. As a cellular pH indicator, Ecliptic (EcGFP) is particularly well-suited to the physiological pH range (pK(a) = 7.2 +/- 0.2) compared with Sapphire (H9; pK(a) = 5.7 +/- 0.1) and a number of other GFPs. However, both EcGFP and H9 exhibit complex, intensity- and wavelength-dependent partitioning between bright and dark EcGFP states, which may complicate their use in quantitative intracellular measurements. Furthermore, the protonation and deprotonation rate constants for the EcGFP chromophore may limit the ability to resolve pH jumps occurring on time scales faster than similar to0.5 ms. A proposed kinetic model of three-state transitions describes quantitatively the interstate conversion via reversible internal and external protonation. The excited neutral state fluorescence lifetime of EcGFP decays is significantly longer (i.e., larger fluorescence quantum yield) than most neutral GFPs, which can be exploited as a new spectral window in biological studies with multiple labels. The pH independence of the EcGFP neutral fluorescence lifetimes implies a ground-state interconversion mechanism for the observed fluorescence quenching in acidic environment.