title: Single-fluorophore biosensors based on conformation-sensitive GFP variants.
abstract: 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.
authors: 2NPA UMR7102, UPMC, 9 quai St. Bernard, 75005 Paris, France. bertrand.lambolez@snv.jussieu.fr.
authors: Agnès
authors: A
authors: Bonnot
authors: Elvire
authors: E
authors: Guiot
authors: Régine
authors: R
authors: Hepp
authors: Laetitia
authors: L
authors: Cavellini
authors: Ludovic
authors: L
authors: Tricoire
authors: Bertrand
authors: B
authors: Lambolez
mesh_terms: D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium
keywords: chromophore; fluorescence lifetime imaging; genetically encoded indicators; green fluorescent protein; high-resolution microscopy; intracellular second messengers