Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature
Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature
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DOI：
10.1021/jp0448595
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发表时间：
2005-07-07
影响因子：
3.3
通讯作者：
Boxer, SG
中科院分区：
文献类型：
--
作者：
Mauring, K;Deich, J;Boxer, SG
携带Y 66 H突变的绿色荧光蛋白表现出强烈的蓝移荧光激发和发射光谱。然而，这些蓝色荧光蛋白（BFP）具有较低的荧光量子产率（Phi（f）类似于0.20），这被认为是源于较小发色团的构象自由度增加。我们证明，通过增加静水压力或降低温度来抑制发色团的迁移率可以提高这些蛋白质的荧光量子产率，而不会显着影响它们的吸收特性或荧光光谱的形状。在皮秒和纳秒制度的荧光寿命的分析表明，荧光量子产率的增强是由于快速猝灭过程的抑制。依赖于温度的荧光测量揭示了两个障碍（类似于19和3千焦/摩尔，分别）的过渡到nonfluorescing状态。这些步骤可能与发色团和His 148或居间水分子之间的氢键的解离以及发色团在激发态中扭曲的屏障有关。在室温下，生色团的氢键平衡由熵效应主导，而在低于200 K时，平衡由熵驱动。
Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) similar to 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore. We demonstrate that suppression of chromophore mobility by increasing hydrostatic pressure or by decreasing temperature can enhance the fluorescence quantum yield of these proteins without significantly affecting their absorption properties or the shape of the fluorescence spectra. Analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes. Temperature-dependent fluorescence measurements reveal two barriers (similar to 19 and 3 kJ/mol, respectively) for the transition into nonfluorescing states. These steps are probably linked with dissociation of the hydrogen bond between the chromophore and His148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. The chromophore's hydrogen-bond equilibrium at room temperature is dominated by entropic effects, while below similar to 200 K the balance is enthalpy-driven.