Zhuo You, Solntsev Kyril M, Reddish Florence, Tang Shen, Yang Jenny J
Department of Chemistry, Georgia State University , Atlanta, Georgia 30303, United States.
J Phys Chem B. 2015 Feb 12;119(6):2103-11. doi: 10.1021/jp501707n. Epub 2014 Jun 5.
We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ.
我们之前设计了一种钙传感器CatchER（一种基于绿色荧光蛋白的钙传感器，用于在诸如内质网等高钙浓度环境中检测高浓度钙离子），它能够监测各种类型细胞中的钙离子反应。钙离子与CatchER结合会引起吸收光谱的比例变化，以及在395和488 nm激发时510 nm处荧光发射增强。在此，我们应用稳态和时间分辨光学方法以及氢/氘同位素交换的组合，以了解CatchER这种钙诱导光学性质变化的起源。我们首先证明，钙离子结合导致间接激发的阴离子发色团的平均荧光寿命增加44%。因此，CatchER是首个具有单一荧光部分的基于蛋白质的钙指示剂，可显示寿命与钙离子结合之间的直接相关性。在质子（相对于重质子）介质中，钙离子对激发态质子转移非绝热双分子复合有强烈抑制作用。对CatchER晶体结构的分析和分子动力学模拟揭示了质子转移机制，即CatchER中被破坏的质子迁移路径通过钙离子结合得以恢复。我们的发现为设计钙传感器的策略提供了重要见解，并表明CatchER可能是用于原位钙荧光寿命成像的有用探针。
