Single-fluorophore biosensors based on conformation-sensitive GFP variants
Résumé
The -strands of GFP form a rigid barrel that protects the chromophore from external influence. Herein, we identified specific mutations in -strand 7 that render the chromophore sensitive to interactions of GFP with another protein domain. In the process of converting the FRET-based protein kinase A (PKA) sensor AKAR2 into a single-wavelength PKA sensor containing a GFP and a quencher, we discovered that the quencher was not required and that the sensor response relied on changes in GFP intrinsic fluorescence. The identified mutations in -strand 7 render GFP fluorescence intensity and lifetime sensitive to conformational changes of the PKA-sensing domain. In addition, sensors engineered from the GCaMP2 calcium indicator to incorporate a conformation-sensitive GFP (csGFP) exhibited calcium-dependent fluorescence changes. We further demonstrate that single GFP sensors report PKA dynamics in dendritic spines of neurons from brain slices on 2-photon imaging with a high signal-to-baseline ratio and minimal photobleaching. The susceptibility of GFP variants to dynamic interactions with other protein domains provides a new approach to generate single wavelength biosensors for high-resolution imaging.Bonnot, A., Guiot, E., Hepp, R., Cavellini, L., Tricoire, L., Lambolez, B. Single-fluorophore biosensors based on conformation-sensitive GFP variants.