Mauring Koit, Deich Jason, Rosell Federico I, McAnaney Tim B, Moerner W E, Boxer Steven G
Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
J Phys Chem B. 2005 Jul 7;109(26):12976-81. doi: 10.1021/jp0448595.
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) approximately 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 ( approximately 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 approximately 200 K the balance is enthalpy-driven.
携带Y66H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）的荧光量子产率较低（Φ(f)约为0.20），据信这源于较小发色团构象自由度的增加。我们证明，通过增加静水压力或降低温度来抑制发色团的流动性，可以提高这些蛋白的荧光量子产率，而不会显著影响其吸收特性或荧光光谱的形状。对皮秒和纳秒时间尺度下荧光寿命的分析表明，荧光量子产率的提高是由于快速猝灭过程受到抑制。温度依赖性荧光测量揭示了两个转变为非荧光状态的能垒（分别约为19和3 kJ/mol）。这些步骤可能分别与发色团和His148或中间水分子之间氢键的解离以及激发态下发色团扭曲的能垒有关。发色团在室温下的氢键平衡主要受熵效应支配，而在约200 K以下，平衡由焓驱动。
