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Electric field stimulates production of highly conductive microbial OmcZ nanowires

Nature Chemical Biology volume 16, pages 1136–1142 (2020)Cite this article

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Abstract

Multifunctional living materials are attractive due to their powerful ability to self-repair and replicate. However, most natural materials lack electronic functionality. Here we show that an electric field, applied to electricity-producing Geobacter sulfurreducens biofilms, stimulates production of cytochrome OmcZ nanowires with 1,000-fold higher conductivity (30 S cm−1) and threefold higher stiffness (1.5 GPa) than the cytochrome OmcS nanowires that are important in natural environments. Using chemical imaging-based multimodal nanospectroscopy, we correlate protein structure with function and observe pH-induced conformational switching to β-sheets in individual nanowires, which increases their stiffness and conductivity by 100-fold due to enhanced π-stacking of heme groups; this was further confirmed by computational modeling and bulk spectroscopic studies. These nanowires can transduce mechanical and chemical stimuli into electrical signals to perform sensing, synthesis and energy production. These findings of biologically produced, highly conductive protein nanowires may help to guide the development of seamless, bidirectional interfaces between biological and electronic systems.

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Fig. 1: Electric field stimulates production of OmcZ nanowires.
Fig. 2: IR nanospectroscopy confirms OmcZ nanowires in biofilms grown under electric field.
Fig. 3: OmcZ nanowires show 1,000-fold higher conductivity than OmcS nanowires.
Fig. 4: OmcZ nanowires show improved π-stacking between hemes versus OmcS and protonation enhances π-stacking.
Fig. 5: Nanoscale IR spectroscopy establishes pH-induced structural transition in OmcS and OmcZ nanowires.
Fig. 6: Raman, CD and fluorescence spectroscopy further demonstrates pH-induced structural transition in OmcS and OmcZ nanowire-containing WT and W51W57 samples, respectively.

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The datasets generated during and/or analyzed during the current study are contained in the published article (and its supplementary information), or available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

The codes used during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank D. Lovley (University of Massachusetts, Amherst) and K. Inoue (University of Miyazaki) for providing strains and OmcZ antibody as well as E. Yan, E. Martz, F. Samatey, C. Salgueiro, C. Shipps and Y. Xiong for helpful discussions. We also thank C. Leang for providing the protocol for immunogold labeling, T. Gokus from Neaspec for help with nanoscale IR imaging and M. Shahid Mansuri, J. Kanyo and T. Lam for help with mass-spectrometry analysis. A portion of the research was performed using Environmental Molecular Sciences Laboratory (EMSL, Ringgold ID 130367), a Department of Energy (DOE) Office of Science User Facility sponsored by the Office of Biological and Environmental Research. S.E.Y. thanks M. Raschke, J. Atkin and S. Lea for help with building the IR s-SNOM setup and C. Smallwood for help with bacteriorhodopsin sample preparation. We thank T. Walsh from Asylum Research for help with stiffness measurements. At Yale, we thank W. Gray from C. Jacobs-Wagner’s laboratory for help with fluorescence microscopy and Z. Wu from H. Wang’s laboratory for help with Raman studies. Computational work was supported by the Air Force Office of Scientific Research Grant FA9550-17-0198 (V.S.B.) and high-performance computing time from the National Energy Research Scientific Computing Center and from the high-performance computing facilities at Yale as well as supercomputer time from the Extreme Science and Engineering Discovery Environment under grant no. TG-CHE170024 (A.A.). Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through Grant R01GM116961 from the National Institutes of Health (NIH). The Anton 2 machine at PSC was generously made available by D.E. Shaw Research. This research was supported by the Career Award at the Scientific Interfaces from Burroughs Welcome Fund (N.S.M.), the NIH Director’s New Innovator award no. 1DP2AI138259-01 (N.S.M.), the National Science Foundation (NSF) CAREER award no. 1749662 (N.S.M.). Research was sponsored by the Defense Advanced Research Project Agency Army Research Office and was accomplished under Cooperative Agreement Number W911NF-18-2-0100 (N.S.M. and V.S.B). This research was supported by NSF Graduate Research Fellowship awards 2017224445 (J.P.O.). Research in the laboratory is also supported by the Charles H. Hood Foundation Child Health Research Award (N.S.M.) and The Hartwell Foundation Individual Biomedical Research Award (N.S.M.).

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Author notes

  1. These authors contributed equally: Sibel Ebru Yalcin, J. Patrick O’Brien.

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Sibel Ebru Yalcin, J. Patrick O’Brien, Sophia M. Yi, Ruchi Jain, Vishok Srikanth, Peter J. Dahl, Dennis Vu & Nikhil S. Malvankar

  2. Microbial Sciences Institute, Yale University, New Haven, CT, USA

    Sibel Ebru Yalcin, J. Patrick O’Brien, Yangqi Gu, Sophia M. Yi, Ruchi Jain, Vishok Srikanth, Peter J. Dahl, Winston Huynh, Dennis Vu & Nikhil S. Malvankar

  3. Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT, USA

    Yangqi Gu

  4. Department of Chemistry, Yale University, New Haven, CT, USA

    Krystle Reiss, Atanu Acharya, Subhajyoti Chaudhuri & Victor S. Batista

  5. Department of Biomedical Engineering, Yale University, New Haven, CT, USA

    Winston Huynh

  6. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA

    Tamas Varga

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Contributions

S.E.Y. and N.S.M. conceived and designed the study. S.E.Y. built the quantum cascade laser-coupled IR s-SNOM detection interferometer, prepared samples, performed AFM, IR s-SNOM measurements, imaged immunogold-labeled nanowires with AFM along with imaging and analysis of reduction in nanowire diameter. J.P.O. grew biofilms in microbial fuel cell and analyzed protein content with R.J. K.R. built the OmcZ model and performed simulations, with help from P.J.D., under the guidance of V.S.B. J.P.O. and W.H. purified nanowires from bacteria, performed CD experiments and conducted analysis. J.P.O. performed FTIR and fluorescence emission spectroscopy and analyzed data with S.E.Y. W.H. and S.E.Y. performed principal component analysis on the IR s-SNOM data. V.S. and Y.G. imaged immunogold-labeled nanowires with TEM. Y.G. also carried out CP–AFM measurements and analyzed data with P.J.D. Y.G. and S.E.Y. performed and analyzed nanowire stiffness measurements. S.M.Y. carried out mass spectroscopy as well as Raman spectroscopy and analyzed Raman data with S.E.Y. Electrode fabrication using electron-beam lithography was carried out by D.V. and Y.G. S.E.Y. and T.V. performed XRD measurements and analyzed data with Y.G. A.A. and S.C. performed initial molecular dynamics simulations under the guidance of V.S.B. A.A. constructed the models, coded the analysis scripts and performed the analysis of molecular dynamics data. N.S.M. supervised the project. S.E.Y. and N.S.M. cowrote the manuscript with input from all authors.

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Correspondence to Sibel Ebru Yalcin or Nikhil S. Malvankar.

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Extended data

Extended Data Fig. 1 Multimodal imaging and nanospectoscopy platform to determine the structure of individual OmcZ and OmcS nanowires as well as their electrical and mechanical properties.

a, Multimodal platform with OmcS nanowire structure showing stacked hemes providing electron transport path (PDB ID: 6EF8). b, c, OmcZ nanowires produced by ΔomcS strain grown under conditions that overexpress OmcZ. b, AFM images of OmcZ nanowires of ΔomcS strain. c, Zoomed image of OmcZ nanowire shown in white square in b. d, AFM Height profile of the OmcZ nanowire taken at the a location shown by a red line in c. Scale bars: b, 100 nm c, 20 nm.

Extended Data Fig. 2 Mass spectrometry and immunoblotting of filament preparations confirm electric field induces overexpression of OmcZ in biofilms.

a, b, Strategy to evaluate the effect of an electric field on the production of OmcZ nanowires. Wild-type G. sulfurreducens cells were grown with a continuous supply of fumarate on graphite electrodes as anodes in a microbial fuel cell. a, An electric field was supplied during the growth of current-producing biofilms by connecting anode to cathode via a potentiostat. b, The electric-field was absent after disconnecting the anodes from the cathodes. OmcZ peptide coverage (blue) in filament preparations of c, Wild-type (WT) and d, W51W57 strain confirming the presence of an extracellular (30 kDa) form of OmcZ that forms nanowires. Comparison of OmcZ abundance in filament preparations of WT and W51W57 strains using e, Immunoblotting and f, Mass spectrometry showing higher level of OmcZ in W51W57 strain than WT. Data represent mean ± standard deviation (n = 3 biologically independent samples overlaid as black circles).

Source data

Extended Data Fig. 3 Immunogold labeling confirms the identity of OmcZ nanowires.

a, AFM image of immunogold-labelled OmcZ nanowire. b, heights of nanowire (red) and gold nanoparticle (blue) at locations shown in a. c–f, TEM images of OmcZ nanowires of ZKI strain in the c, absence of OmcZ antibody and d, in the presence of anti-OmcZ antibody. Secondary antibody with gold nanoparticles was used in both c–e, No OmcZ labelling was found for filaments of ΔomcZ strain f, Labelling for OmcZ nanowires of W51W57 strain. Scale bars, a, 100 nm, c, 50 nm, d, 25 nm, e, 100 nm, f, 25 nm.

Extended Data Fig. 4 Low pH reduces the diameter of individual OmcS and OmcZ nanowires.

Histograms of AFM measurements of OmcS and OmcZ nanowire heights showed that lowering the pH reduced the diameter of a, OmcS and b, OmcZ nanowires. Values represent mean ± standard deviation (s.d.) (n = 100 measurements of nanowires over 3 biologically independent samples).

Source data

Extended Data Fig. 5 IR s-SNOM imaging of bacteriorhodopsin (bR) confirms α-helical structure.

a, b Schematic of IR s-SNOM. a, The interferometer is comprised of a tunable quantum cascade laser (QCL) for tip illumination, a beam splitter (BS), a detector (Mercury Cadmium Telluride, MCT), a parabolic mirror (PM), and a reference mirror. b, Schematic of the IR s-SNOM setup used for bR imaging (PDB ID: 1m0l). All helices are parallel to electric field lines that enhance the amide I signal. c, AFM topography and corresponding height profile for bR taken at a location shown by a black line. d, IR s-SNOM near-field phase (absorption) images for bR at various IR excitations. At 1660 cm-1 (iii, on-resonant IR), the amide I absorption is enhanced in the near-field phase data. However, when changed to other frequencies (i-ii & iv-v, off-resonance), the phase signal decreases and drops to zero. e, Spatiospectral analysis of near-field phase data for the amide I mode of bacteriorhodopsin. The blue line corresponds to a fit of the imaginary part of a Lorentzian (Lor) with peak at 1663 cm−1 and line width of 25 cm−1. Data represent mean ± standard deviation for individual bR proteins (n = 3 biologically independent samples).

Source data

Extended Data Fig. 6 Bulk FTIR and IR s-SNOM confirm lysozyme structure and IR s-SNOM spectroscopy of OmcS nanowires agrees with Cryo-EM structure.

Multi-peak fitting function was used to fit the data using a, Gaussian profile for bulk FTIR and b, a Lorentzian profile for IR s-SNOM data. α-helix corresponds to 1662 cm−1, β-sheet corresponds to 1618 cm−1 and 1678 cm−1, and the loop (D) region corresponds to 1635 cm−1. c, Schematic of the IR s-SNOM setup for nanowire imaging. Secondary structure of the OmcS nanowire at pH 10.5 is shown in a (PDB ID: 6EF8) with α-helices in red, 310 helices in pink and beta strands in green. d, At pH 10.5 used to solve the structure of OmcS nanowires, spatiospectral analysis of near-field phase data of the amide I mode of OmcS nanowire. The blue line corresponds to a fit of imaginary parts of a Lorentzian, with peak positions at 1669 cm−1 and 1643 cm−1 corresponding to α-helical and loop regions respectively. Data represent mean ± standard deviation for individual OmcS nanowires (n = 3 biologically independent samples).

Source data

Extended Data Fig. 7 Model of OmcZ structure reveals highly stacked hemes and beta sheets in agreement with experiments.

a, Computational model of OmcZ. b, Superposition of hemes from five 8-heme cytochromes (PDB ID:4QO5:Green, 1FGJ:Blue, 3GM6:Cyan, 6H5L:Gray, and 6HIF:Pink). Both c, computed and d, experimental cryo-EM density for OmcZ show similar shape and size. e-h, Consecutive histidines cause tight heme T-junction. The His-His binding motif found in OmcZ also exists in c3 cytochromes, (PDB ID: e, 1gyo, f, 2bq4, and g, 2e84). In OmcZ model (h), the three pairs of adjacent histidines that bind to T-stacked hemes are all distal histidine ligands bound opposite to the proximal histidine in the CXXCH sequence. In the c3 cytochromes, all the heme-binding adjacent histidine pairs are mixed, containing both a proximal and a distal histidine. Despite this difference, the distance between the heme pairs for c3 and OmcZ is similar (~6.0 Å).

Extended Data Fig. 8 Reduction in nanowire diameter enhances their stiffness.

AFM topography, Young’s modulus, and the stiffness distribution for OmcS nanowires at a, pH 7 and b, pH 2 and for OmcZ nanowires c, pH 7 and d, pH 2. Scale bars: a and d, 100 nm; b and c, 200 nm. Values represent mean ± (s. d.) for n = 512 ×512 measurements of nanowires over three biologically independent samples.

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Extended Data Fig. 9 Bulk FTIR spectra of OmcS and OmcZ nanowires show transition to β-sheets at pH 2 and conformation change is independent of buffers.

FTIR spectra at pH 7 and pH 2 for a, OmcS nanowires and b, OmcZ nanowires showing a red shift, consistent with transition to β-sheets. c, Water does not contribute to the amide I spectra because OmcS nanowires at pH 7 under air-dried and D2O conditions display similar spectra. d, e, OmcS nanowires at pH 7 and pH 2 in 10 mM Potassium Phosphate and 20 mM Citrate buffer characterized by d, Solution CD spectra and e, Solid-state CD spectra. f, FTIR spectra for OmcS nanowires in Citrate buffer at pH 2 showing a red shift, consistent with transition to β-sheets. g, Representative AFM image and h, corresponding height profile at a location shown by a red line in g for OmcS nanowires in citrate buffer at pH 2 under air-dried conditions. Scale bar, 200 nm.

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Extended Data Fig. 10 Purified OmcS and OmcZ nanowires from ΔomcZ and KN400 strains respectively show conformational change to β-sheets similar to nanowires of wild-type and W51W57 strains.

SDS-PAGE gel of filament preparations showing a single band corresponding to a, OmcS purified from ΔomcZ strain and c, OmcZ from KN400 strain. Corresponding TEM images of b, OmcS and e, OmcZ nanowires. Scale bars, 400 nm. Solution CD spectra of c, OmcS and f, OmcZ is similar to CD spectra of OmcS and OmcZ nanowires purified from wild-type and W51W57 strains respectively (Fig. 6c, d). M1 and M2 represent nanowires sheared from cells by two different methods – vortexing (M1) and blending (M2).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Tables 1 and 2.

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Supplementary Data

PDB file for OmcZ model

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Yalcin, S.E., O’Brien, J.P., Gu, Y. et al. Electric field stimulates production of highly conductive microbial OmcZ nanowires. Nat Chem Biol 16, 1136–1142 (2020). https://doi.org/10.1038/s41589-020-0623-9

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