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Single‐fluorophore biosensors based on conformation‐sensitive GFP variants
Author(s) -
Bonnot Agnès,
Guiot Elvire,
Hepp Régine,
Cavellini Laetitia,
Tricoire Ludovic,
Lambolez Bertrand
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.13-240507
Subject(s) - green fluorescent protein , photobleaching , fluorophore , förster resonance energy transfer , biophysics , chromophore , fluorescence , chemistry , biosensor , mutant , biochemistry , biology , photochemistry , physics , quantum mechanics , gene
The β‐strands of GFP form a rigid barrel that protects the chromophore from external influence. Herein, we identified specific mutations in β‐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 β‐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. FASEB J. 28, 1375–1385 (2014). www.fasebj.org