Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

Michael Russwurm , Florian Mullershausen , Andreas Friebe , Ronald Jäger , Corina Russwurm , Doris Koesling (1)

1 Institut für Pharmakologie und Toxikologie

The intracellular signalling molecule cGMP regulates a variety of physiological processes; hence monitoring cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create fluorescence resonance energy transfer (FRET)-based cGMP indicators from the two known types of cGMP-binding domains, cNMP-BD and GAF, found in cGMP-dependent protein kinase and phosphodiesterase 5, respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive, however, the GAF-derived sensors had to be discarded because of slow kinetics. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with half maximally effective concentrations between 500 nM and 6 µM. These indicators feature excellent cGMP specificity, fast kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of the new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics measured by radioimmunoassays.

Mots clés: Life Sciences

Michael Russwurm, Florian Mullershausen, Andreas Friebe, Ronald Jäger, Corina Russwurm, et al.. Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach. Biochemical Journal, 2007, 407 (1), pp.69-77. ⟨10.1042/BJ20070348⟩. ⟨hal-00478771⟩