Fig. 1: Development and in vitro characterization of R-eLACCO2. | Nature Communications

Fig. 1: Development and in vitro characterization of R-eLACCO2.

From: A red fluorescent genetically encoded biosensor for in vivo imaging of extracellular l-lactate dynamics

Fig. 1

a Schematic representation of R-eLACCO and its mechanism of response to l-lactate. b Schematic of directed evolution workflow. c ΔF/F rank plot representing all proteins tested during the directed evolution. d Excitation and emission spectra of R-eLACCO2 in the presence (10 mM) and absence of l-lactate. e Dose-response curves of R-eLACCO2, R-eLACCO2.1, and R-deLACCOctrl for l-lactate. n = 3 experimental triplicates (mean ± s.d.). f Absorbance spectra of R-eLACCO2 in the presence (50 mM) and absence of l-lactate. g Fluorescence image of a streak plate of E. coli expressing R-eLACCO1 or R-eLACCO2. h Two-photon excitation spectra of R-eLACCO2 in the presence (10 mM) and absence of l-lactate. ΔF/F is represented in the green plots. GM, Goeppert-Mayer units. i Dose-response curve of R-eLACCO2 as a function of Ca2+ in various l-lactate concentrations. Fluorescence intensities were normalized based on the largest fluorescence value in the graph. n = 3 experimental triplicates (mean ± s.d.). j pH titration curves of R-eLACCO2 in various l-lactate concentrations. Fluorescence intensities were normalized based on the largest fluorescence value in the graph. n = 3 experimental triplicates (mean ± s.d.). k Specificity of R-eLACCO2. Concentration of each metabolite is 10 mM. Right graph represents a dose-response curve of R-eLACCO2 for D-lactate. n = 3 experimental triplicates (mean ± s.d.). One-way analysis of variance (ANOVA) with the Dunnett’s post hoc tests. ***p < 0.0001. ***p (Dunnett’s, each metabolite vs. l-lactate as a control group) <0.0001. l Dose-lifetime curves (left) and Δτ (right) of R-eLACCO2, R-eLACCO2.1, and R-deLACCOctrl for l-lactate. n = 3 experimental triplicates (mean ± s.d.). m pH-lifetime curves of R-eLACCO2 in the presence (100 mM) and absence of l-lactate. n = 3 experimental triplicates (mean ± s.d.). n Cryo-EM structure of R-eLACCO2 in the lactate bound state. l-Lactate and Ca2+ (black) are shown in sphere representations. o Density map and constructed model of the chromophore. The two “gate post” residues (Val194 and Trp433)13 in cpmApple of R-eLACCO2 are highlighted in dark orange and purple, respectively. Source data are provided as a Source Data file.

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