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Determination of Copper(II) Ion Concentration by Lifetime Measurements of Green Fluorescent Protein

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Abstract

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 His6-tag as the responsible binding site for Cu2 +  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 [Cu2 + ] which is comprehended by a mathematical titration model. We envision that Cu2 +  can be quantified noninvasively and in real-time by measuring τ fl of GFP.

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Acknowledgements

We are indebted to D. Auerbach for help with the expression and N. Baltes for careful reading of the manuscript. The authors also thank T. Seidel (Bielefeld) for kindly providing us with the eYFP-plasmid. This work was supported by German Research Foundation (DFG, grant JU 650/2-1 and 2-2).

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Authors and Affiliations

  1. Department of Biophysical Chemistry, Saarland University, Campus B2 2, 66123, Saarbrücken, Germany

    Benjamin Hötzer, Silke Altmeier & Gregor Jung

  2. Department of Biosciences-Plant Biology, Saarland University, Campus A2 4, 66123, Saarbrücken, Germany

    Rumen Ivanov

  3. Department of Biophysics, Saarland University, Building 76, 66421, Homburg, Germany

    Reinhard Kappl

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  1. Benjamin Hötzer
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  2. Rumen Ivanov
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  3. Silke Altmeier
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  5. Gregor Jung
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Correspondence to Gregor Jung.

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Hötzer, B., Ivanov, R., Altmeier, S. et al. Determination of Copper(II) Ion Concentration by Lifetime Measurements of Green Fluorescent Protein. J Fluoresc 21, 2143–2153 (2011). https://doi.org/10.1007/s10895-011-0916-1

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  • DOI: https://doi.org/10.1007/s10895-011-0916-1

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