Effect of Ca²⁺ on the steady-state and time-resolved emission properties of the genetically encoded fluorescent sensor CatchER.
Effect of Ca²⁺ on the steady-state and time-resolved emission properties of the genetically encoded fluorescent sensor CatchER.
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DOI：
10.1021/jp501707n
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发表时间：
2015-02-12
期刊：
影响因子：
--
通讯作者：
Yang JJ
中科院分区：
文献类型：
--
作者：
Zhuo Y;Solntsev KM;Reddish F;Tang S;Yang JJ
我们先前设计了钙传感器CatchER（用于在高钙浓度环境（例如ER）中检测高浓度的基于GFP的钙传感器），其具有监测各种类型细胞中的钙离子响应的能力。钙与CatchER结合诱导吸收光谱的比率变化，以及在395和488 nm激发时在510 nm处的荧光发射增加。在这里，我们已经应用了稳态和时间分辨光学方法和氢/氘同位素交换的组合，以了解钙诱导的光学性质变化的CatchER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是第一个基于蛋白质的钙指示剂，具有单个荧光部分，显示寿命和钙结合之间的直接相关性。钙对质子（相对于氘）介质中激发态质子转移的非绝热孪生复合有很强的抑制作用。CatchER晶体结构的分析和MD模拟揭示了质子转移机制，其中CatchER中被破坏的质子迁移路径被钙结合拯救。我们的发现为设计钙传感器的策略提供了重要的见解，并表明CatchER可能是原位钙的FLIM成像的有用探针。
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.
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