Exploration of fluorescent protein voltage probes based on circularly permuted fluorescent proteins.
Exploration of fluorescent protein voltage probes based on circularly permuted fluorescent proteins.
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DOI：
10.3389/neuro.16.014.2009
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Knöpfel T
中科院分区：
文献类型：
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作者：
Gautam SG;Perron A;Mutoh H;Knöpfel T
基因编码的荧光蛋白（FP）电压传感器是很有前途的工具，用于光学监测的电活动的细胞。在过去的十年中，已经探索了几种融合蛋白的设计，并且其中一些已经被证明足够灵敏以记录来自单个哺乳动物细胞的膜电压瞬变。最突出的是电压敏感荧光蛋白（VSFP）家族，其利用了玻璃海鞘含电压传感器的磷酸酶（Ci-VSP）的电压传感器结构域（VSD）。这些先前报道的膜电位指示剂的荧光读出的电压灵敏度通过两个FP光谱变体之间的荧光共振能量转移效率的变化或通过单个FP的荧光强度的调制来实现。在这里，我们报告我们的探索第三VSFP设计原则的基础上循环排列的荧光蛋白（cpFP）的变种。使用来自GCaMP 2的环状排列的EGFP和两个新产生的命名为mKate的远红外发射蛋白的环状排列的变体，我们产生并表征了一系列电压敏感探针，其中cpFP与Ci-VSP的VSD融合。最有希望的变体是基于环状排列的mKate，其具有由残基180和182给出的新的N-和C-末端。尽管如此，它们的电压灵敏度相对适中，但它们构成了这种新型蛋白质设计的原理证明。
Genetically encoded fluorescent protein (FP) voltage sensors are promising tools for optical monitoring of the electrical activity of cells. Over the last decade, several designs of fusion proteins have been explored and some of them have proven to be sensitive enough to record membrane voltage transients from single mammalian cells. Most prominent are the families of voltage sensitive fluorescent proteins (VSFPs) that utilize the voltage sensor domain (VSD) of Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP). The voltage sensitivity of the fluorescence readout of these previously reported membrane potential indicators is achieved either via a change in the efficiency of fluorescence resonance energy transfer between two FP spectral variants or via modulation in the fluorescence intensity of a single FP. Here, we report our exploration on a third VSFP design principle based on circularly permuted fluorescent protein (cpFP) variants. Using circularly permuted EGFP derived from GCaMP2 and two newly generated circularly permuted variants of the far-red emitting protein named mKate, we generated and characterized a series of voltage-sensitive probes wherein the cpFPs were fused to the VSD of Ci-VSP. The most promising variants were based on circularly permuted mKate with new N- and C-termini given by residues 180 and 182. Even so their voltage sensitivity was relatively modest, they constitute a proof of principle for this novel protein design.