Determination of Copper(II) Ion Concentration by Lifetime Measurements of Green Fluorescent Protein
Determination of Copper(II) Ion Concentration by Lifetime Measurements of Green Fluorescent Protein
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DOI：
10.1007/s10895-011-0916-1
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发表时间：
2011-11-01
影响因子：
2.7
通讯作者：
Jung, Gregor
中科院分区：
文献类型：
--
作者：
Hoetzer, Benjamin;Ivanov, Rumen;Jung, Gregor
了解细胞的过程和功能并阐明其生理机制是生命科学的一个重要目标。一个重要的方面是必需物质的摄取和释放，以及它们与细胞环境的相互作用。由于绿色荧光蛋白(GFP)可以在细胞中进行遗传编码，因此它可以作为内部传感器，从而更深入地了解生化途径。在这里，我们报道了铜离子的存在导致了绿色荧光蛋白的荧光寿命(tau(Fl))的缩短，并为Forster共振能量转移(FRET)作为猝灭机制提供了证据。我们确定His(6)-Tag是Cu2+的主要结合部位，其解离常数K(D)=9+/-2µM，Forster半径R(0)=2.1+/-0.1 nm。寿命猝灭的程度取决于[Cu2+]，这可以用数学滴定模型来理解。我们设想，通过测量绿色荧光蛋白的tau(Fl)，可以非侵入性地实时定量Cu2+。
The understanding of cellular processes and functions and the elucidation of their physiological mechanisms is an important aim in the life sciences. One important aspect is the uptake and the release of essential substances as well as their interactions with the cellular environment. As green fluorescent protein (GFP) can be genetically encoded in cells it can be used as an internal sensor giving a deeper insight into biochemical pathways. Here we report that the presence of copper(II) ions leads to a decrease of the fluorescence lifetime (tau (fl)) of GFP and provide evidence for Forster resonance energy transfer (FRET) as the responsible quenching mechanism. We identify the His(6)-tag as the responsible binding site for Cu2 + with a dissociation constant K (d) = 9 +/- 2 mu M and a Forster radius R (0) = 2.1 +/- 0.1 nm. The extent of the lifetime quenching depends on [Cu2 + ] which is comprehended by a mathematical titration model. We envision that Cu2 + can be quantified noninvasively and in real-time by measuring tau (fl) of GFP.