1). Single-fluorophore biosensors based on conformation-sensitive GFP variants. Alvimopan monohydrate Keywords:green fluorescent protein, chromophore, genetically encoded indicators, intracellular second messengers, fluorescence lifetime imaging, high-resolution microscopy The chromophore of the green fluorescent protein (GFP) fromAequorea victoriaessentially results from autocyclization of the SYG[6567]residues buried inside the rigid barrel-shaped structure of the protein (1). Nonetheless, other amino acid residues of the protein contribute to its optical properties. Accordingly, spectral variants of GFP have been obtained through amino acid substitutions in several regions of the protein, such as the popular blue-shifted cyan fluorescent protein (CFP; ref.2) and yellow-shifted yellow fluorescent protein (YFP; ref.3) variants, as well as a dark YFP (4) endowed with YFP absorption properties but minimal emission intensity. A large variety of genetically encoded biosensors use GFP and its variants to report conformational changes of specific sensing domains elicited by biological signals (5,6). Engineering of these sensors has been mainly based on two strategies. One relies on changes of fluorescence resonance energy transfer (FRET) between two spectrally different fluorescent proteins (7). A similar paradigm uses the dark-YFP mutant to quench the emission of a fluorescent protein on conformational changes of the sensor (4,8). In principle, this latter paradigm circumvents the need for spectral separation and allows the collection of photons in the entire emission spectrum. The other strategy is based on the modulation of optical properties of a single, circularly permuted, fluorescent protein (5). This approach has yielded single-wavelength sensors with high signal-to-baseline responses but whose low baseline fluorescence often hampers visualization of sensor-expressing cells in tissue slices (911). Here, we used a dark-YFP-based quenching approach to engineer a sensor aimed at reporting protein kinase A (PKA) activity in brain slices using 2-photon microscopy. The ubiquitous cyclic AMP/PKA pathway is a key intracellular signal pathway. Cyclic adenosine monophosphate (cAMP) is produced by adenylate cyclase and activates PKA, which exerts pleiotropic effects in the cell. In the brain, the cAMP/PKA pathway modulates neuronal excitability and synaptic transmission and mediates the effects of multiple neurotransmitters. The PKA FRET sensor A-kinase activity reporter 2 (AKAR2; UNG2 ref.12) contains a sensing domain, formed with a phosphothreonine binding domain and a PKA substrate peptide, flanked by CFP and YFP (seeFig. 1). Alvimopan monohydrate When phosphorylated by PKA, the substrate peptide folds into the phosphothreonine binding pocket, which causes an increase of the YFP/CFP emission ratio. == Figure Alvimopan monohydrate 1. == Constructs derived from the FRET sensor AKAR2.A) Mechanisms of fluorescence changes reported for AKAR2 and anticipated for the newly generated GAKdY sensor following folding of the substrate peptide (SP) into the Forkhead-associated domain (FHA) on phosphorylation.B) Domain structure of most constructs used in this study, named after their constituent parts (see Abbreviations). Color boxes, gray boxes, and white boxes represent active fluorophores, fluorophores converted into quenchers, and inactivated fluorophores, respectively. Mutations at positions 6567 (chromophore) and dark-YFP mutations are indicated. Inactivation of the sensing domain has been achievedviamutagenesis in SP (crossed box). Here, we mutated the CFP and YFP domains of AKAR2 into GFP and the dark-YFP quencher, respectively. The new sensor reported PKA activation in living cells by an increase in fluorescence intensity. To explore the mechanisms of these fluorescence changes, additional sensors were engineered by inactivation, deletion, or substitution of the chromophores. We found that the interaction between two fluorophore domains was not required to generate a responsive sensor and that specific amino acid substitutions inside -strand 7 of GFP were instead critical. We confirmed this property of -strand 7 mutant of GFP on other sensors engineered from an existing calcium indicator. Finally, we found that PKA sensors incorporating this specific GFP mutant as single fluorophore exhibited a redistribution of fluorescence lifetimes on PKA activation and allowed the imaging of PKA dynamics in brain slices with high spatial resolution. == MATERIALS AND METHODS == == PKA and calcium sensors == The PKA activity reporter AKAR2.1 (12) comprising the enhanced CFP (2) and the citrine version of YFP (13) was subcloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA). Constructs were derived from AKAR2.1 by site-directed mutagenesis (Quikchange XL kit, Stratagene, La Jolla, CA, USA) or fluorophore deletion and substitution as described inFig. 1andTable 1. A subset of these constructs (seeTable.