The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 μM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.
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Research Article|
September 12 2007
Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach
Michael Russwurm
;
Michael Russwurm
1
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Federal Republic of Germany
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Florian Mullershausen
;
Florian Mullershausen
1
1
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Federal Republic of Germany
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Andreas Friebe
;
Andreas Friebe
1
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Federal Republic of Germany
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Ronald Jäger
;
Ronald Jäger
1
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Federal Republic of Germany
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Corina Russwurm
;
Corina Russwurm
1
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Federal Republic of Germany
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Doris Koesling
Doris Koesling
2
1
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Federal Republic of Germany2
To whom correspondence should be addressed (doris.koesling@ruhr-uni-bochum.de).
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Biochem J (2007) 407 (1): 69-77.
Article history
Received:
March 12 2007
Revision Received:
May 16 2007
Accepted:
May 22 2007
Accepted Manuscript online:
May 22 2007
Citation
Michael Russwurm, Florian Mullershausen, Andreas Friebe, Ronald Jäger, Corina Russwurm, Doris Koesling; Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach. Biochem J 1 October 2007; 407 (1): 69–77. doi: https://doi.org/10.1042/BJ20070348
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