![Figure 2. The genetically encoded family of biosensors for nicotinic drugs, iNicSnFRs. (A) Cartoon of the x-ray crystallographic structure of iNicSnFR1, crystallized in the presence of nicotine. The structure is available as PDB file 6EFR. The iNiCSnFR family are fusion proteins. A superfolder cpGFP (shown in green) has been inserted into the coding sequence of OpuBC, a choline/betaine PBP from T. spX513. The linker sequences (shown in dark blue; see Fig. S1) were selected for optimal ΔF/F. One poorly resolved linker residue, Pro323, is shown as a dashed backbone. The engineered OpuBC is shown in cyan, except that the backbone residues near the incompletely resolved nicotine ligand are shown in gray. The nicotine-binding site lies between the two lobes of the PBP; these move relative to each other. (B) To generate later iNicSnFRs, the binding site of OpuBC was further engineered by mutagenesis for acceptable sensitivity to nicotine. ∼12,000 mutants were screened during the design of iNicSnFR3a and iNicSnFR3b. The image shows redesign of the nicotine-binding site of iNicSnFR1, naming the additional mutations present in iNicSnFR3a and iNicSnFR3b. Portions of the cartoon shown in gray are identical to the gray regions of OpuBC in A. The α-carbon atoms remain at the positions in the x-ray crystallographic data (PDB file 6EFR), and the conformers of the mutated side chains were selected based on the best-fit rotamer using University of California, San Francisco (San Francisco, CA) Chimera software. The pyrrolidine group of nicotine is at left, seen edge-on; the pyridine group is at right, seen from an acute angle. Also shown is Y357, which remains wild type in all iAChSnFR and iNicSnFR constructs (Fig. S1). The Y357A mutation renders all iNicSnFR and iAChSnFR constructs insensitive to the ligand. Also shown is F391, which remains unchanged in all iNicSnFR and iAChSnFR constructs, but differs from the glutamate in OpuBC. (C) Dose–response relations for purified iNicSnFR3a. Data were fitted to a single Hill equation with an assumed Hill coefficient of 1. Data are mean ± SEM (n ≤ 3). (D) Stopped-flow analysis. The rate constant for fluorescence decay, koff, was measured most consistently by extrapolation of the kon value to zero [nicotine] (koff-ext). The ratio, koff-ext/kon, gives an equilibrium-binding constant Kd = 29 µM. The blue symbols and label show measurements of koff in experiments that diluted an iNicSnFR3a-nicotine solution by 25-fold to [nicotine] values < 1 µM (koff-dil). (E) Isothermal titration calorimetry for nicotine. The data yield an equilibrium-binding constant Kd = 10 ± 2.5 µM, and a stoichiometry of 0.65 ± 0.03 moles of nicotine per mole of protein.](https://cdn.rupress.org/rup/content_public/journal/jgp/151/6/10.1085_jgp.201812201/9/jgp_201812201_fig2.jpeg?Expires=1773807580&Signature=LJh~IsbT8HzUIwCwBOiXTmwDKlyPbH-ZuUvzjPs41cdYmzI9-r43JwIraKFJ-voLSsiHyULNXdPBvus5oDsSE3Aw0m8rtsj~fEP7Xm2tKyt893RYfYv3XW54V-Di~ZOfcBjjOMpvVs37ZAYqJ2DvbTMRieE5MdEio1Ij7obnX9np6~zBbLyMKZrUD9yXOy7PbC8VzgMffCbCSeZtt7Lytj6hAOZG07Hk1vj8B2MM2uI0AOgOG0lIBcJd8lEOKTsHt7xXT2-9seL1RQ4ByCeu3PfXY4tiiEHWyI3VQxAPV3w6b32pIxymqI-0fD5ySC3ZGRJLRXYseVcd9NwTCpXF6w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The genetically encoded family of biosensors for nicotinic drugs, iNicSnFRs. (A) Cartoon of the x-ray crystallographic structure of iNicSnFR1, crystallized in the presence of nicotine. The structure is available as PDB file 6EFR. The iNiCSnFR family are fusion proteins. A superfolder cpGFP (shown in green) has been inserted into the coding sequence of OpuBC, a choline/betaine PBP from T. spX513. The linker sequences (shown in dark blue; see Fig. S1) were selected for optimal ΔF/F. One poorly resolved linker residue, Pro323, is shown as a dashed backbone. The engineered OpuBC is shown in cyan, except that the backbone residues near the incompletely resolved nicotine ligand are shown in gray. The nicotine-binding site lies between the two lobes of the PBP; these move relative to each other. (B) To generate later iNicSnFRs, the binding site of OpuBC was further engineered by mutagenesis for acceptable sensitivity to nicotine. ∼12,000 mutants were screened during the design of iNicSnFR3a and iNicSnFR3b. The image shows redesign of the nicotine-binding site of iNicSnFR1, naming the additional mutations present in iNicSnFR3a and iNicSnFR3b. Portions of the cartoon shown in gray are identical to the gray regions of OpuBC in A. The α-carbon atoms remain at the positions in the x-ray crystallographic data (PDB file 6EFR), and the conformers of the mutated side chains were selected based on the best-fit rotamer using University of California, San Francisco (San Francisco, CA) Chimera software. The pyrrolidine group of nicotine is at left, seen edge-on; the pyridine group is at right, seen from an acute angle. Also shown is Y357, which remains wild type in all iAChSnFR and iNicSnFR constructs (Fig. S1). The Y357A mutation renders all iNicSnFR and iAChSnFR constructs insensitive to the ligand. Also shown is F391, which remains unchanged in all iNicSnFR and iAChSnFR constructs, but differs from the glutamate in OpuBC. (C) Dose–response relations for purified iNicSnFR3a. Data were fitted to a single Hill equation with an assumed Hill coefficient of 1. Data are mean ± SEM (n ≤ 3). (D) Stopped-flow analysis. The rate constant for fluorescence decay, koff, was measured most consistently by extrapolation of the kon value to zero [nicotine] (koff-ext). The ratio, koff-ext/kon, gives an equilibrium-binding constant Kd = 29 µM. The blue symbols and label show measurements of koff in experiments that diluted an iNicSnFR3a-nicotine solution by 25-fold to [nicotine] values < 1 µM (koff-dil). (E) Isothermal titration calorimetry for nicotine. The data yield an equilibrium-binding constant Kd = 10 ± 2.5 µM, and a stoichiometry of 0.65 ± 0.03 moles of nicotine per mole of protein.
