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Metalloprotein switches that display chemical-dependent electron transfer in cells


Biological electron transfer is challenging to directly regulate using environmental conditions. To enable dynamic, protein-level control over energy flow in metabolic systems for synthetic biology and bioelectronics, we created ferredoxin logic gates that utilize transcriptional and post-translational inputs to control energy flow through a synthetic electron transfer pathway that is required for bacterial growth. These logic gates were created by subjecting a thermostable, plant-type ferredoxin to backbone fission and fusing the resulting fragments to a pair of proteins that self-associate, a pair of proteins whose association is stabilized by a small molecule, and to the termini of a ligand-binding domain. We show that the latter domain insertion design strategy yields an allosteric ferredoxin switch that acquires an oxygen-tolerant [2Fe–2S] cluster and can use different chemicals, including a therapeutic drug and an environmental pollutant, to control the production of a reduced metabolite in Escherichia coli and cell lysates.

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Fig. 1: Designing split Fds that transfer electrons within cells.
Fig. 2: Post-translational control over Fd electron transfer in cells.
Fig. 3: Using domain insertion to create a Fd switch.
Fig. 4: Using purified sFd-35-ER to control metabolite production in cell lysates.

Data availability

There are no restrictions on data availability. Accession codes and sequences used for multiple sequence alignments are provided in Supplementary Note 1. Raw data for Figs. 1, 2 and 3 are available upon request.


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E. coli EW11 and the genes encoding Zea mays FNR, Zea mays SIR, and Spinacia oleracea Fd were a gift from P. Silver (Harvard University). Cellular assay development was supported by DOE grant DE-SC0014462 (G.N.B. and J.J.S.), split Fd efforts were supported by NASA grant NNX15AL28G (J.J.S. and G.N.B.), domain insertion efforts were supported by ONR grant N00014-17-1-2639 (J.J.S.), and electrochemistry was supported by DOE grant DE-SC0012598 (S.J.E.). J.T.A. was supported by NSF GRFP and DOE SGCSR fellowships.

Author information




J.T.A. designed and constructed all DNA vectors, performed the multiple sequence analysis, and did all cellular experiments. I.J.C. purified proteins and performed the lysate experiments. E.E.T. evaluated the switch substrate specificity profile. S.C.B. and S.J.E. performed the voltammetry. J.T.A., J.J.S., and G.N.B. conceptualized the project. J.T.A. and J.J.S. wrote the manuscript.

Corresponding author

Correspondence to Jonathan J. Silberg.

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Competing interests

J.J.S., J.T.A., G.N.B., and I.J.C. have submitted a patent application (No 16/186,226) covering the use of fragmented Fds as chemical-dependent electron carriers, entitled “Regulating electron flow using fragmented proteins.”

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Supplementary information

Supplementary Text and Figures

Supplementary Table 1, Supplementary Figures 1–9

Reporting Summary

Supplementary Note 1

Full-length multiple structure/sequence alignment of plant-type Fds used for sequence divergence profile calculation

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Atkinson, J.T., Campbell, I.J., Thomas, E.E. et al. Metalloprotein switches that display chemical-dependent electron transfer in cells. Nat Chem Biol 15, 189–195 (2019).

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