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The HaloTag as a general scaffold for far-red tunable chemigenetic indicators

Nature Chemical Biology volume 17, pages 718–723 (2021)Cite this article

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Abstract

Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for ‘chemigenetic’ fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

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Fig. 1: Strategy for engineering chemigenetic indicators from HaloTag.
Fig. 2: HaloCaMP engineering and in vitro characterization.
Fig. 3: Synthesis of Si-rhodamine derivatives and characterization of HaloCaMP in neuron cultures.
Fig. 4: Chemigenetic voltage indicators.

Data availability

Plasmids have been deposited at Addgene (www.addgene.org) as follows: pAAV-synapsin-HaloCaMP1a (plasmid 138327), pAAV-synapsin-HaloCaMP1b (plasmid 138328), pCAG-HASAP1 (plasmid 138325) and pCAG-HArcLight1 (plasmid 138326). Crystal structures of HaloTag–TMR and HaloCaMP1b635 have been deposited at the Protein Data Bank (www.rcsb.org) with accession codes 6U32 and 6U2M, respectively. Source data from experiments in this study are available from the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We acknowledge the Molecular Biology, Cell Culture and Virus Production facilities at Janelia for assistance. This work was supported by the Howard Hughes Medical Institute. B.M. holds a postdoctoral fellowship from the Research Foundation–Flanders (FWO Vlaanderen). The Berkeley Center for Structural Biology is supported in part by the Howard Hughes Medical Institute. The Advanced Light Source is a Department of Energy Office of Science User Facility under contract no. DE-AC02-05CH11231.

Author information

Author notes

  1. Claire Deo

    Present address: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

  2. Hersh K. Bhargava

    Present address: Biophysics Graduate Program, University of California, San Francisco, San Francisco, CA, USA

  3. Natalie Falco

    Present address: Pharmacological Sciences Graduate Program, University of California, Irvine, Irvine, CA, USA

  4. Benjamien Moeyaert

    Present address: Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium

  5. These authors contributed equally: Claire Deo, Ahmed S. Abdelfattah.

  6. These authors jointly supervised this work: Luke D. Lavis, Eric R. Schreiter.

Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Claire Deo, Ahmed S. Abdelfattah, Hersh K. Bhargava, Adam J. Berro, Natalie Falco, Helen Farrants, Benjamien Moeyaert, Mariam Chupanova, Luke D. Lavis & Eric R. Schreiter

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Contributions

C.D., A.S.A., L.D.L. and E.R.S. conceived the project and wrote the manuscript. C.D. contributed organic synthesis, protein engineering, in vitro characterization of proteins, X-ray crystallography and neuron imaging. A.S.A. contributed protein engineering, neuron imaging and electrophysiology. H.K.B. contributed protein engineering, in vitro characterization and neuron imaging. A.J.B. contributed organic synthesis and X-ray crystallography. N.F. contributed organic synthesis. H.F. measured stopped-flow kinetics of purified HaloCaMP. B.M. and M.C. contributed protein engineering and neuron imaging. L.D.L. and E.R.S. directed the project.

Corresponding authors

Correspondence to Luke D. Lavis or Eric R. Schreiter.

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Competing interests

C.D., A.S.A., H.K.B., L.D.L. and E.R.S. have filed patent applications on chemigenetic indicators and azetidine-substituted fluorophores.

Additional information

Peer review information Nature Chemical Biology thanks Adam Cohen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Multiple conformations of tetramethylrhodamine (TMR) in the HaloTag-TMR structure (PDB 6U32).

Stereo view of the region of the bound dye ligand with the HaloTag protein backbone represented as grey cartoon, Asp106 and the bound HaloTag ligand are shown as sticks with grey carbon atoms, and the TMR dye is modeled in two conformations, represented as sticks with black or white carbon atoms. The TMR conformation with black carbon atoms is the one represented in Fig. 1e,f. The 2fo-fc electron density map for the bound dye ligand is shown as purple mesh, contoured at 0.8σ. Although the electron density is strong and continuous for the HaloTag ligand, density for the dye moiety is weaker and broken, suggesting conformational heterogeneity.

Extended Data Fig. 2 Exploration of circular permutation sites within HaloTag for sensor design.

a, Crystal structure of HaloTag (grey cartoon ribbons) bound to TMR–HaloTag ligand (sticks), illustrating circular permutation sites tested as green spheres labeled with the amino acid number. b, Schematic of the sensor context for testing HaloTag circular permutations, with cpHaloTag inserted between helices 3 and 4 of a VSD (top), the field stimulus pattern for stimulation of neurons expressing voltage sensors with cpHaloTag variants (middle), and representative fluorescence traces of each voltage sensor variant in stimulated neurons (bottom).

Source data

Extended Data Fig. 3 Synthesis of Si-rhodamine derivatives.

Synthesis routes and yields of azetidine-substituted Si-rhodamine derivatives (a) and their corresponding HaloTag ligands (b).

Extended Data Fig. 4 Spectroscopy of azetidine-substituted Si-rhodamine derivatives.

a, Absorption, (b) normalized excitation and (c) normalized fluorescence emission spectra of novel azetidine-substituted Si-rhodamines. d, Correlation of experimental λex versus inductive Hammett constants (σΙ) for Si-rhodamines. e, Normalized absorption (bold line) and fluorescence (dashed line) spectra of Si-rhodamine HaloTag ligands, in the presence (colored line) or absence (black line) of HaloTag protein. Values were normalized to the HaloTag-bound spectra. All spectra were measured at C = 5 µM in 10 mM HEPES pH = 7.4. In the case of the HaloTag ligands, 0.1 mg.mL-1 CHAPS was added to the buffer.

Source data

Extended Data Fig. 5 Calcium titrations.

Solution calcium titration curves for purified HaloCaMP1a (a) and HaloCaMP1b (b) labeled with Si-rhodamine ligands.

Source data

Extended Data Fig. 6 Labeling of HaloCaMP in neuron cultures.

Cultured hippocampal neurons expressing HaloCaMP1a-GFP (a) or HaloCaMP1b-GFP labeled with with JF635-HTL (1 μM, 30 min). GFP channel (left panel), JF635 channel (middle panel) and merge (right panel); scale bars: 50 μm.

Extended Data Fig. 7 Comparison of HaloCaMP with NIR-GECO1.

a, Comparison of the relative fluorescence brightness (product of quantum yield and extinction coefficient) measured in purified protein solutions. b-d, Measurements in primary neuron cultures with field stimulation-driven action potentials of (b) maximum ΔF/F0 (c) fluorescence rise time, and (d) fluorescence decay time.

Source data

Extended Data Fig. 8 Fluorescence response in neuron culture of the initial variant of HASAP loaded with JF635-HTL.

Black triangles denote the timing of electrical stimuli from the field electrode. Compare with Fig. 4i after additions of the R467G mutation.

Source data

Extended Data Fig. 9 Fluorescence vs. Voltage for HASAP1635.

Fluorescence traces of HASAP1635 in response to a series of voltage steps (from -110 mV to +50 mV in 20 mV increments). Image acquisition rate = 400 Hz. Representative traces from one cell of N = 8 cells for HASAP1635.

Source data

Extended Data Fig. 10 Fluorescence vs. Voltage for HArcLight1635.

Fluorescence traces of HArcLight1635 in response to a series of voltage steps (from -110 mV to +50 mV in 20 mV increments). Image acquisition rate = 400 Hz. Representative traces from one cell of N = 9 cells.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Tables 1–6 and Supplementary Notes.

Reporting Summary

Supplementary Video 1

Raw fluorescence video, captured at 400 Hz and displayed in real time, of a rat hippocampal neuron in culture expressing HASAP1 and labeled with JF635–HaloTag ligand. Voltage is clamped at −70 mV and stepped using a patch electrode to +30 mV for 1 s.

Source data

Source Data Fig. 1

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Source Data Extended Data Fig. 2

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Deo, C., Abdelfattah, A.S., Bhargava, H.K. et al. The HaloTag as a general scaffold for far-red tunable chemigenetic indicators. Nat Chem Biol 17, 718–723 (2021). https://doi.org/10.1038/s41589-021-00775-w

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