Jung Gregor, Wiehler Jens, Zumbusch Andreas
Department Chemie and Center for Nanoscience, LMU Munich, Munich, Germany.
Biophys J. 2005 Mar;88(3):1932-47. doi: 10.1529/biophysj.104.044412. Epub 2004 Dec 21.
The three amino acids S65, T203, and E222 crucially determine the photophysical behavior of wild-type green fluorescent protein. We investigate the impact of four point mutations at these positions and their respective combinations on green fluorescent protein's photophysics using absorption spectroscopy, as well as steady-state and time-resolved fluorescence spectroscopy. Our results highlight the influence of the protein's hydrogen-bonding network on the equilibrium between the different chromophore states and on the efficiency of the excited-state proton transfer. The mutagenic approach allows us to separate different mechanisms responsible for fluorescence quenching, some of which were previously discussed theoretically. Our results will be useful for the development of new strategies for the generation of autofluorescent proteins with specific photophysical properties. One example presented here is a variant exhibiting uncommon blue fluorescence.
氨基酸S65、T203和E222对野生型绿色荧光蛋白的光物理行为起着关键决定作用。我们利用吸收光谱以及稳态和时间分辨荧光光谱，研究了这些位置的四个点突变及其各自组合对绿色荧光蛋白光物理性质的影响。我们的结果突出了蛋白质氢键网络对不同发色团状态之间平衡以及激发态质子转移效率的影响。诱变方法使我们能够区分导致荧光猝灭的不同机制，其中一些机制此前已有理论探讨。我们的结果将有助于开发新策略，以产生具有特定光物理性质的自发荧光蛋白。本文给出的一个例子是一种呈现罕见蓝色荧光的变体。
