Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction

Abstract

Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding "core" that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a Kd of ∼14 µM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue.

Keywords: Förster resonance energy transfer (FRET); ancestral protein reconstruction; biosensor; fluorescence; neurobiology; nitric oxide; protein engineering.

Figure 1

Reconstruction of ancestral polar amino acid PBPs. Midpoint-rooted maximum likelihood phylogeny of the amino acid-binding protein family used for reconstruction of ancestral sequences. Branches are drawn to scale and labeled with bootstrap values from 100 replicates. Sequences are identified by their GI number from the NCBI database, and are labeled with their putative binding specificity on the basis of functional annotation and homology to proteins of known function. The scale bar represents 0.4 substitutions per site. Three clades of closely related proteins are compressed for clarity.

Figure 2

Characterization of the reconstructed ancestral proteins AncQ and AncQR. (A) SDS-PAGE gel showing overexpression of AncQ, AncQR, and seArgT in soluble fractions of E. coli crude cell lysates. Cultures were grown at 37°C in Terrific Broth media overnight. Samples were run on an ExpressPlus 4 to 20% PAGE gel (GenScript) and stained with Coomassie Blue. (B) Thermal unfolding of AncQ, AncQR and seArgT as monitored by CD spectroscopy. CD was measured at 222 nm. The T_m of seArgT was determined to be 51.6 ± 0.7°C (mean ± SD, n = 4) by fitting the data to a two-state denaturation model, where θ = CD (mdeg), T = temperature (°C), ΔH = enthalpy of unfolding (J/mol), R = gas constant (J/K/mol), T_m = denaturation temperature (°C), and θ = m₁T + b₁ and θ = m₂T + b₂ give the pretransition and post-transition baselines, respectively. (C) Baseline-corrected power traces resulting from continuous injection isothermal titration calorimetry experiments, illustrating binding of l-arginine, l-glutamine, l-histidine, and l-lysine by AncQ and AncQR. Amino acids (750 µM) were injected continuously into protein (50 µM) between t = 120 s and t = 420 s. Injection of other proteinogenic amino acids resulted in negligible deviations in power from baseline levels. (D) Binding specificity of AncQ. The K_d of AncQ for l-arginine, l-lysine, l-ornithine, l-histidine, and l-glutamine was determined by ITC. These experiments were performed in 50 mM Na₂HPO₄ pH 7.40, 100 mM NaCl buffer at 25°C. Error bars represent standard deviation of the mean from at least two separate titrations.

Figure 3

Characterization of FLIPR and cpFLIPR. A) Fluorescence spectra of FLIPR (turquoise and blue) and cpFLIPR (crimson and red) with peaks at 475 nm (CFP) and 525 nm (Venus) indicating change in fluorescence ratio upon addition of saturating ligand. FLIPR does not exhibit any change in ratio with addition of 1 mM l-arginine, while cpFLIPR maximally shows a 35% change in ratio. B and C) PHYRE2 models of AncQ (B) and cpAncQ (C). N & C terminal regions are indicated by the blue and red secondary structures respectively, with spheres displaying the termini. The circular permutation is illustrated by the removal of the second hinge and the relocation of the termini (C). D) Sigmoidal dose response curves for cpFLIPR with l-arginine (red), l-lysine (orange), l-histidine (purple), l-ornithine (green), and l-glutamine (blue). Values are the (525 nm/475 nm) fluorescence ratio as a percentage of the same ratio for the apoenzyme. Curves were fitted using the following equation.. Values are the mean ± standard error (n = 3).

Figure 4

Comparison of stability between cpFLIPR and contemporary sensor cpArtJ (20). A) Fluorescence ratio (%, relative to apo) as a function of temperature. cpArtJ (apo: dark blue, 1 mM Arg: light blue) indicates a strong temperature dependence and lower stability than cpFLIPR (apo: crimson, 1 mM Arg: red). The data were fitted using a Boltzmann function ( where B = Y min, T = Y max, V50 = Y mid. B) Ratio changes of cpArtJ (blue) and cpFLIPR (red) upon incubation at 37°C for 45 min normalized (as percentages) against the aposensor ratio. Pre incubation measurements were conducted as separate experiments. cpArtJ displays a diminished VFP/CFP ratio at 37°C postincubation and this change is irreversible as shown by the postincubation 25°C ratio, which is equivalent to that of the aposensor. All changes are statistically significant (P < 0.05) as determined by a t-test (preincubation relative to postincubation measurements) and a paired t-test (postincubation measurements).

Figure 5

Characterization and in situ testing of cpFLIPR using two-photon excitation (2PE) fluorescence microscopy. A) Calibration curve of cpFLIPR using 2PE shows a dose-dependent VFP/CFP fluorescence ratio change. Fitting the relationship between fluorescence ratio R and l-arginine concentration c with a Hill equation obtains a K_d of 14.04 ± 3.8 µM, n = 3. B) Schematic representation of sensor immobilization in brain tissue. Biotinylated cpFLIPR was conjugated to streptavidin (SA, gray) and then puffed into brain slices preincubated with EZ-link Biotin leading to its attachment to cell surfaces (brown). C) Illustrates the puff of cpFLIPR (yellow) into the striatum radiatum of the CA1 region of a rat hippocampal slice at a depth of ∼70 µm. The highlighted region was subsequently imaged at a depth of 59 µm. D) cpFLIPR responses to application of l-arginine in situ demonstrate its capability to measure steady state conditions and sensitivity to changes in ligand concentration in organized brain tissues. Fluorescence ratios at rest and after application of 50 µM and 1 mM of l-arginine were used to calculate the resting l-arginine concentration in acute brain slices.