Abstract
For the quantitative analysis of molecular processes in living (plant) cells, such as the perception and processing of environmental and endogenous signals, new combinatorial approaches in optical and spectroscopic technologies are required and partly already became established in many fields of the life sciences. One hallmark of the in vivo analysis of cell biological processes is the use of visible fluorescent proteins to create fluorescent fusion proteins. Recent progress has been made in generating a redox-sensitive mutant of green fluorescent proteins (roGFP), which exhibits alterations in its spectral properties in response to changes in the redox state of the surrounding medium. An established method to probe the local redox potential using roGFP is based on a ratiometric protocol. This readout modality requires two excitation wavelengths, which makes the technique less suited for in vivo studies of e.g. dynamic samples. We clarify the origin of the redox sensitivity of roGFP by ab initio calculations, which reveal a changed protonation equilibrium of the chromophore in dependence on the redox potential. Based on this finding, we test and compare different spectroscopic readout modalities with single wavelength excitation to determine the local redox potential and apply these techniques to live cell analytics.
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Acknowledgments
We thank Andreas J. Meyer, University of Bonn, Germany, for providing the roGFP2 construct and the seeds of the cytoplasmic Grx1-roGFP line. This study was supported by the DFG (FOR948:ZE 313/8-1) and the Land Baden-Württemberg.
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Wierer, S., Peter, S., Elgass, K. et al. Determination of the in vivo redox potential by one-wavelength spectro-microscopy of roGFP. Anal Bioanal Chem 403, 737–744 (2012). https://doi.org/10.1007/s00216-012-5911-0
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DOI: https://doi.org/10.1007/s00216-012-5911-0