An expandable, modular de novo protein platform for precision redox engineering

George H. Hutchins; Claire E.M. Noble; H. Adrian Bunzel; Christopher Williams; Paulina Dubiel; Sathish K.N. Yadav; Paul M. Molinaro; Rob Barringer; Hector Blackburn; Benjamin J. Hardy; Alice E. Parnell; Charles Landau; Paul R. Race; Thomas A A Oliver; Ronald L. Koder; Matthew P. Crump; Christiane Schaffitzel; A. Sofia F. Oliveira; Adrian J. Mulholland; J. L.Ross Anderson

doi:10.1073/pnas.2306046120

An expandable, modular de novo protein platform for precision redox engineering

George H. Hutchins, Claire E.M. Noble, H. Adrian Bunzel, Christopher Williams, Paulina Dubiel, Sathish K.N. Yadav, Paul M. Molinaro, Rob Barringer, Hector Blackburn, Benjamin J. Hardy, Alice E. Parnell, Charles Landau, Paul R. Race, Thomas A A Oliver, Ronald L. Koder, Matthew P. Crump, Christiane Schaffitzel, A. Sofia F. Oliveira, Adrian J. Mulholland, J. L.Ross Anderson^*

^*Corresponding author for this work

Research output: Contribution to journal › Article (Academic Journal) › peer-review

5 Citations (Scopus)

Abstract

The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

Original language	English
Article number	e2306046120
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	120
Issue number	31
Early online date	24 Jul 2023
DOIs	https://doi.org/10.1073/pnas.2306046120 https://doi.org/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10400981/
Publication status	Published - 1 Aug 2023

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS. This work was supported at the University of Bristol by the Biological and Biotechnological Sciences Research Council (BBW003449/1, BB/ R016445/1, BB/M02315X/1, BB/M025624/1, BB/M009122/1 & BB/T008741/1, the latter two providing a studentship for G.H.H.) and the SynBioCDT (EPSRC and BBSRC Centre for Doctoral Training in Synthetic Biology Grant EP/L016494/1) for studentships for C.E.M.N., B.J.H., and P.D. This work was also supported at City College New York by the NSF (MCB-2025200). This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation programme (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M., C.S. acknowledges funding from the Wellcome Trust (210701/Z/18/Z). A.S.F.O. is an Oracle for Research Fellow (with A.J.M.). The Authors also wish to thank Oracle for Research for providing Cloud Time. MD simulations were carried out using the computational facilities of the Advanced Computing Research Centre,University of Bristol (http://bris.ac.uk/acrc/). We would also like to thank Dr.Peter Wilson in the School of Biochemistry Biosuite at the University of Bristol for access to biophysical equipment and Dr.Ufuk Borucu for assistance with cryo-EM data collection. We acknowledge support and assistance by the Wolfson Bioimaging Facility and the GW4 Facility for High-Resolution Electron Cryo-Microscopy funded by the Wellcome Trust (202804/Z/16/Z and 206181/Z/17/Z) and BBSRC (BB/R000484/1).We also wish to thank the Diamond light Source staff, in particular those who support beamline I03.

Publisher Copyright:
Copyright © 2023 the Author(s).

Structured keywords

Bristol BioDesign Institute
BrisSynBio

Keywords

bioenergetics
heme proteins
protein design

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10400981/Licence: CC BY

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Hutchins, G. H., Noble, C. E. M., Bunzel, H. A., Williams, C., Dubiel, P., Yadav, S. K. N., Molinaro, P. M., Barringer, R., Blackburn, H., Hardy, B. J., Parnell, A. E., Landau, C., Race, P. R., Oliver, T. A. A., Koder, R. L., Crump, M. P., Schaffitzel, C., Oliveira, A. S. F., Mulholland, A. J., & Anderson, J. L. R. (2023). An expandable, modular de novo protein platform for precision redox engineering. Proceedings of the National Academy of Sciences of the United States of America, 120(31), Article e2306046120. https://doi.org/10.1073/pnas.2306046120, https://doi.org/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10400981/

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title = "An expandable, modular de novo protein platform for precision redox engineering",

abstract = "The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme {"}maquette{"} protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.",

keywords = "bioenergetics, heme proteins, protein design",

author = "Hutchins, {George H.} and Noble, {Claire E.M.} and Bunzel, {H. Adrian} and Christopher Williams and Paulina Dubiel and Yadav, {Sathish K.N.} and Molinaro, {Paul M.} and Rob Barringer and Hector Blackburn and Hardy, {Benjamin J.} and Parnell, {Alice E.} and Charles Landau and Race, {Paul R.} and Oliver, {Thomas A A} and Koder, {Ronald L.} and Crump, {Matthew P.} and Christiane Schaffitzel and Oliveira, {A. Sofia F.} and Mulholland, {Adrian J.} and Anderson, {J. L.Ross}",

note = "Funding Information: ACKNOWLEDGMENTS. This work was supported at the University of Bristol by the Biological and Biotechnological Sciences Research Council (BBW003449/1, BB/ R016445/1, BB/M02315X/1, BB/M025624/1, BB/M009122/1 & BB/T008741/1, the latter two providing a studentship for G.H.H.) and the SynBioCDT (EPSRC and BBSRC Centre for Doctoral Training in Synthetic Biology Grant EP/L016494/1) for studentships for C.E.M.N., B.J.H., and P.D. This work was also supported at City College New York by the NSF (MCB-2025200). This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation programme (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M., C.S. acknowledges funding from the Wellcome Trust (210701/Z/18/Z). A.S.F.O. is an Oracle for Research Fellow (with A.J.M.). The Authors also wish to thank Oracle for Research for providing Cloud Time. MD simulations were carried out using the computational facilities of the Advanced Computing Research Centre,University of Bristol (http://bris.ac.uk/acrc/). We would also like to thank Dr.Peter Wilson in the School of Biochemistry Biosuite at the University of Bristol for access to biophysical equipment and Dr.Ufuk Borucu for assistance with cryo-EM data collection. We acknowledge support and assistance by the Wolfson Bioimaging Facility and the GW4 Facility for High-Resolution Electron Cryo-Microscopy funded by the Wellcome Trust (202804/Z/16/Z and 206181/Z/17/Z) and BBSRC (BB/R000484/1).We also wish to thank the Diamond light Source staff, in particular those who support beamline I03. Publisher Copyright: Copyright {\textcopyright} 2023 the Author(s).",

year = "2023",

month = aug,

day = "1",

doi = "10.1073/pnas.2306046120",

language = "English",

volume = "120",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

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Hutchins, GH, Noble, CEM, Bunzel, HA, Williams, C , Dubiel, P , Yadav, SKN, Molinaro, PM, Barringer, R, Blackburn, H, Hardy, BJ, Parnell, AE, Landau, C, Race, PR, Oliver, TAA, Koder, RL, Crump, MP , Schaffitzel, C , Oliveira, ASF , Mulholland, AJ & Anderson, JLR 2023, 'An expandable, modular de novo protein platform for precision redox engineering', Proceedings of the National Academy of Sciences of the United States of America, vol. 120, no. 31, e2306046120. https://doi.org/10.1073/pnas.2306046120, https://doi.org/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10400981/

TY - JOUR

T1 - An expandable, modular de novo protein platform for precision redox engineering

AU - Hutchins, George H.

AU - Noble, Claire E.M.

AU - Bunzel, H. Adrian

AU - Williams, Christopher

AU - Dubiel, Paulina

AU - Yadav, Sathish K.N.

AU - Molinaro, Paul M.

AU - Barringer, Rob

AU - Blackburn, Hector

AU - Hardy, Benjamin J.

AU - Parnell, Alice E.

AU - Landau, Charles

AU - Race, Paul R.

AU - Oliver, Thomas A A

AU - Koder, Ronald L.

AU - Crump, Matthew P.

AU - Schaffitzel, Christiane

AU - Oliveira, A. Sofia F.

AU - Mulholland, Adrian J.

AU - Anderson, J. L.Ross

N1 - Funding Information: ACKNOWLEDGMENTS. This work was supported at the University of Bristol by the Biological and Biotechnological Sciences Research Council (BBW003449/1, BB/ R016445/1, BB/M02315X/1, BB/M025624/1, BB/M009122/1 & BB/T008741/1, the latter two providing a studentship for G.H.H.) and the SynBioCDT (EPSRC and BBSRC Centre for Doctoral Training in Synthetic Biology Grant EP/L016494/1) for studentships for C.E.M.N., B.J.H., and P.D. This work was also supported at City College New York by the NSF (MCB-2025200). This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation programme (PREDACTED Advanced Grant Agreement no. 101021207) to A.J.M., C.S. acknowledges funding from the Wellcome Trust (210701/Z/18/Z). A.S.F.O. is an Oracle for Research Fellow (with A.J.M.). The Authors also wish to thank Oracle for Research for providing Cloud Time. MD simulations were carried out using the computational facilities of the Advanced Computing Research Centre,University of Bristol (http://bris.ac.uk/acrc/). We would also like to thank Dr.Peter Wilson in the School of Biochemistry Biosuite at the University of Bristol for access to biophysical equipment and Dr.Ufuk Borucu for assistance with cryo-EM data collection. We acknowledge support and assistance by the Wolfson Bioimaging Facility and the GW4 Facility for High-Resolution Electron Cryo-Microscopy funded by the Wellcome Trust (202804/Z/16/Z and 206181/Z/17/Z) and BBSRC (BB/R000484/1).We also wish to thank the Diamond light Source staff, in particular those who support beamline I03. Publisher Copyright: Copyright © 2023 the Author(s).

PY - 2023/8/1

Y1 - 2023/8/1

N2 - The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

AB - The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

KW - bioenergetics

KW - heme proteins

KW - protein design

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U2 - 10.1073/pnas.2306046120

DO - 10.1073/pnas.2306046120

M3 - Article (Academic Journal)

C2 - 37487099

AN - SCOPUS:85165765943

SN - 0027-8424

VL - 120

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 31

M1 - e2306046120

ER -

Hutchins GH, Noble CEM, Bunzel HA, Williams C , Dubiel P , Yadav SKN et al. An expandable, modular de novo protein platform for precision redox engineering. Proceedings of the National Academy of Sciences of the United States of America. 2023 Aug 1;120(31):e2306046120. Epub 2023 Jul 24. doi: 10.1073/pnas.2306046120, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10400981/

An expandable, modular de novo protein platform for precision redox engineering

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An expandable, modular de novo protein platform for precision redox engineering

Abstract

Bibliographical note

Structured keywords

Keywords

Access to Document

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Other files and links

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HPC (High Performance Computing) and HTC (High Throughput Computing) Facilities

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