Determination of copper(II) ion concentration by lifetime measurements of green fluorescent protein.

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.