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Chemigenetic indicators based on synthetic chelators and green fluorescent protein

Abstract

Molecular fluorescent indicators are versatile tools for dynamic imaging of biological systems. We now report a class of indicators that are based on the chemigenetic combination of a synthetic ion-recognition motif and a protein-based fluorophore. Specifically, we have developed a calcium ion (Ca2+) indicator that is based on genetic insertion of circularly permuted green fluorescent protein into HaloTag protein self-labeled with a ligand containing the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. We have demonstrated the versatility of this design by also developing a sodium ion (Na+) indicator using a crown-ether-containing ligand. This approach affords bright and sensitive ion indicators that can be applicable to cell imaging. This design can enable the development of chemigenetic indicators with ion or molecular specificities that have not been realized with fully protein-based indicators.

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Fig. 1: Strategy for developing the hybrid ion sensors.
Fig. 2: Development and characterization of HaloGFP-Ca14.
Fig. 3: Imaging of dynamic changes in intracellular [Ca2+] with HaloGFP-Ca1–11 and HaloGFP-Ca0.5–11 under two excitation wavelengths.
Fig. 4: Molecular modeling of the HaloGFP-Ca1–4 indicator.
Fig. 5: Development and characterization of HaloGFP-Na0.5–14.

Data availability

The authors declare that all custom software and data supporting the findings of this study are available within the paper and its Supplementary Information or are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank T. Ueno and Y. Urano (Graduate School of Pharmaceutical Sciences, The University of Tokyo) for providing access to their instruments. We thank H. Sato, Y. Hori, and T. Tomita (Graduate School of Pharmaceutical Sciences, The University of Tokyo) for technical advice on rat primary neuron culture. We thank JSPS KAKENHI (19H05633 to R.E.C.; 18H02103 and 21H00273 to T.Terai) and Kato Memorial Bioscience Foundation (to Y.N.) for financial support. W.Z. was supported by the MERIT-WINGS program of The University of Tokyo. T.Terai was also supported by Tokuyama Science Foundation and The Asahi Glass Foundation. T.Terada was supported by Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Numbers JP21am0101107 and JP22ama121027.

Author information

Authors and Affiliations

Authors

Contributions

W.Z. performed all experiments related to HaloGFP-Ca. S.T. performed all experiments related to HaloGFP-Na. S.I. developed the higher-affinity HaloGFP-Ca0.2 variant. T. Terada performed molecular modeling and MD simulation. T.U. performed stopped-flow analysis. R.E.C. conceived of the design of indicator design, and he supervised the project together with T. Terai. R.E.C., T. Terai., Y.N., S.I., S.T., and W.Z. designed experiments and analyzed data related to protein engineering. R.E.C., T. Terai., S.T., and W.Z designed experiments and analyzed data related to synthetic molecules. W.Z., S.T., T. Terai., Y.N., T. Terada, and R.E.C. wrote and edited the manuscript.

Corresponding authors

Correspondence to Takuya Terai or Robert E. Campbell.

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The authors declare no competing interest.

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Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Ca2+-dependent changes in excitation spectra.

Ca2+-dependent changes in excitation spectra (emission wavelength of 555 ± 20 nm) of HaloGFP (residues 143–144 were replaced with GFP) reacted with various 4BAPTA-PEG-Haloligands. Gray line represents the fluorescence intensity in the absence of Ca2+, and the black line represents the fluorescence intensity in the presence of 36 μM free [Ca2+]. (a) Without ligand. (b) Ligand 1. (c) Ligand 2. (d) Ligand 3. (e) Ligand 4. (f) Ligand 5. N = 3 independent experiments (mean ± s.d.).

Source data

Extended Data Fig. 2 Residue positions targeted during interface optimization.

Residue positions targeted during interface optimization, shown in the context of the crystal structures of cpGFP derived from GCaMP6m (PDB ID 3WLC) and HaloTag (PDB ID 5Y2Y). cpGFP structure is in green, HaloTag is in blue, and the rim region of the HaloTag active site is orange. The red amino acids (numbered based on Extended Data Fig. 3) are the bulky and solvent-exposed residues which were manually selected for randomization.

Extended Data Fig. 3 Sequence alignment of HaloTag, cpGFP, and HaloGFP-Ca variants in this work.

Sequence alignment of HaloTag, cpGFP, and HaloGFP-Ca variants in this work. The HaloTag part is in blue and the cpGFP part is in green. Numbers at the top correspond to residue numbering for the full-length indicators. The mutations accumulated during indicator optimization are represented with red (during interface optimization) and black (during random mutagenesis). The extra glycines in gray were added as part of linkers.

Extended Data Fig. 4 In vitro characterization of HaloGFP-Ca variants.

Absorption spectra, fluorescence spectra, Ca2+ titration curves and pH-dependent fluorescence changes for HaloGFP-Ca variants. N = 3 independent experiments (mean ± s.d.).

Source data

Extended Data Fig. 5 Intracellular Ca2+ imaging of HaloGFP-Ca variants (additional data).

Intracellular Ca2+ imaging of HaloGFP-Ca1 fused to either an NLS (nuclear localization signal) or an NES (nuclear export signal) in HeLa cells, and HaloGFP-Ca0.5 in a neuron cell. (a, c) Time course of fluorescence intensities and excitation ratio of HaloTag-Ca1 with an NLS (a) or an NES (c). (e) Time course of fluorescence intensities and excitation ratio of HaloTag-Ca0.5 in a neuron cell. The fluorescence intensity was calculated in the region of interest indicated by the yellow dotted box in (b, d, f). (b, d, f) Fluorescence images were taken at different excitation wavelengths and times as indicated. Scale bar: 10 μm.

Source data

Extended Data Fig. 6 Na+-dependent changes in excitation spectra.

Na+-dependent changes in excitation spectra (emission wavelength of 555 ± 20 nm) of HaloGFP (residues 143–144 were replaced with GFP) reacted with five different lengths of chloroalkane ligand. Fluorescence intensity was measured in buffer solution with [K+] + [Na+] = 400 mM. The gray line represents the fluorescence intensity in the absence of Na+ ([K+] = 400 mM), and the black line represents the fluorescence intensity in the presence of 400 mM [Na+] ([K+] = 0 mM). (a) Without ligand. (b) Ligand 12. (c) Ligand 13. (d) Ligand 14. (e) Ligand 15. (f) Ligand 16.

Source data

Extended Data Fig. 7 Residue positions mutated during optimization of HaloGFP-Na by directed evolution.

Residue positions mutated during optimization of HaloGFP-Na by directed evolution. Structure is represented using cpGFP derived from GCaMP6m (PDB ID 3WLC) and HaloTag (PDB ID 5Y2Y). cpGFP structure is in green, HaloTag is in blue, and the rim region of the HaloTag active site is orange. The gray dashed lines represent the connecting sites of two proteins, and the green dashed line means the linker of the original N- and C-termini of GFP. The residues mutated during random mutagenesis are shown in red. A2T and E537G are also mutated residues but not shown in the figure since they are missing from the published crystal structure.

Extended Data Fig. 8 Sequence alignment of HaloTag, cpGFP, and the HaloGFP-Na variants developed in this work.

Sequence alignment of HaloTag, cpGFP, and the HaloGFP-Na variants developed in this work. The HaloTag part is in blue and the cpGFP part is in green. Numbers at the top correspond to residues numbering for the full-length indicators. The mutations accumulated during indicator optimization are represented with red (during random mutagenesis). The extra glycines in gray were added as a part of linkers.

Extended Data Fig. 9 Absorption and fluorescence emission spectra for HaloGFP-Na variants.

Absorption and fluorescence emission spectra for HaloGFP-Na variants in 30 mM Tris-HCl buffer (pH 7.2) containing either 0 mM Na+ (Na+(–)), 200 mM K+(K+(+)), or 200 mM Na+(Na+(+)).

Source data

Extended Data Fig. 10 pH-dependent fluorescence changes for HaloGFP-Na variants.

pH-dependent fluorescence changes for HaloGFP-Na variants. Samples were in buffer condition (pH 7.2) with either 0 mM Na+ (Na+(–)), 200 mM K+(K+(+)), or 200 mM Na+(Na+(+)). Sample protein concentrations were the same for all variants and normalization was done based on the largest fluorescence intensity of three conditions (Na+(–), K+(+), Na+(+)). N = 3 independent experiments (mean ± s.d.).

Source data

Supplementary information

Supplementary Information

Supplementary Figs 1–11, Supplementary Tables 1–9, synthetic scheme, and NMR spectra.

Reporting Summary

Supplementary Video 1

Fluorescence change of HaloGFP-Ca1 in HeLa cells after stimulation by histamine, Ca2+, and EGTA as shown in Supplementary Fig. 5a–d.

Supplementary Data 1

Source data of Supplementary Fig.1.

Supplementary Data 2

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Supplementary Data 3

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Supplementary Data 6

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Supplementary Data 10

Source data of Supplementary Fig.11.

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Zhu, W., Takeuchi, S., Imai, S. et al. Chemigenetic indicators based on synthetic chelators and green fluorescent protein. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01134-z

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