Department of Biophysical Chemistry, Saarland University, Saarbrücken, Germany.
J Fluoresc. 2011 Nov;21(6):2143-53. doi: 10.1007/s10895-011-0916-1. Epub 2011 Jul 20.
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 (τ(fl)) of GFP and provide evidence for Förster resonance energy transfer (FRET) as the responsible quenching mechanism. We identify the His(6)-tag as the responsible binding site for Cu(2+) with a dissociation constant K(d) = 9 ± 2 μM and a Förster radius R(0) = 2.1 ± 0.1 nm. The extent of the lifetime quenching depends on [Cu(2+)] which is comprehended by a mathematical titration model. We envision that Cu(2+) can be quantified noninvasively and in real-time by measuring τ(fl) of GFP.
细胞过程和功能的理解以及其生理机制的阐明是生命科学的重要目标。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) 可以在细胞内进行基因编码，因此它可以用作内部传感器，深入了解生化途径。在这里，我们报告说铜 (II) 离子的存在会导致 GFP 荧光寿命 (τ(fl)) 的降低，并提供了Förster 共振能量转移 (FRET) 是负责猝灭机制的证据。我们确定 His(6)-标签是 Cu(2+) 的结合位点，解离常数 K(d) = 9 ± 2 μM，Förster 半径 R(0) = 2.1 ± 0.1nm。寿命猝灭的程度取决于 [Cu(2+)]，这可以通过数学滴定模型来理解。我们设想可以通过测量 GFP 的 τ(fl) 来非侵入性和实时地定量 Cu(2+)。
