Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Elementary tetrahelical protein design for diverse oxidoreductase functions

Nature Chemical Biology volume 9, pages 826–833 (2013)Cite this article

Subjects

A Corrigendum to this article was published on 17 January 2014

This article has been updated

Abstract

Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Helical bundle topology and cofactor insertion.
Figure 2: Secondary and tertiary structuring in maquettes.
Figure 3: 750 MHz 15N-HSQC showing changes in NMR spectral dispersion upon heme binding.
Figure 4: Heme spectra and kinetics of substrate binding and electron transfer.
Figure 5: Light activated electron transfer experiments with a protein maquette.

Change history

  • 11 December 2013

    In the version of this article initially published, the US Department of Energy Office of Basic Energy Sciences, Energy Frontier Research Center grant number was incorrect. The correct number is DE-SC 0001035. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Fischer, E. Nobel Lectures, Chemistry 1901–1921 Vol.1 (ed. Nobelstiftelsen) 21–35 (Elsevier, Amsterdam, 1966).

  2. Brustad, E.M. & Arnold, F.H. Optimizing non-natural protein function with directed evolution. Curr. Opin. Chem. Biol. 15, 201–210 (2011).

    Article  CAS  Google Scholar 

  3. Baker, D. An exciting but challenging road ahead for computational enzyme design. Protein Sci. 19, 1817–1819 (2010).

    Article  CAS  Google Scholar 

  4. Prabhulkar, S., Tian, H., Wang, X.T., Zhu, J.J. & Li, C.Z. Engineered proteins: redox properties and their applications. Antioxid. Redox Signal. 17, 1796–1822 (2012).

    Article  CAS  Google Scholar 

  5. Edelman, G.M. & Gally, J.A. Degeneracy and complexity in biological systems. Proc. Natl. Acad. Sci. USA 98, 13763–13768 (2001).

    Article  CAS  Google Scholar 

  6. Lichtenstein, B.R. et al. Engineering oxidoreductases: maquette proteins designed from scratch. Biochem. Soc. Trans. 40, 561–566 (2012).

    Article  CAS  Google Scholar 

  7. Regan, L. & DeGrado, W.F. Characterization of a helical protein designed from first principles. Science 241, 976–978 (1988).

    Article  CAS  Google Scholar 

  8. Page, C.C., Moser, C.C., Chen, X. & Dutton, P.L. Natural engineering principles of electron tunnelling in biological oxidation-reduction. Nature 402, 47–52 (1999).

    Article  CAS  Google Scholar 

  9. Robertson, D.E. et al. Design and synthesis of multi-heme proteins. Nature 368, 425–432 (1994).

    Article  CAS  Google Scholar 

  10. Sharp, R.E., Moser, C.C., Rabanal, F. & Dutton, P.L. Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette. Proc. Natl. Acad. Sci. USA 95, 10465–10470 (1998).

    Article  CAS  Google Scholar 

  11. Shifman, J.M., Moser, C.C., Kalsbeck, W.A., Bocian, D.F. & Dutton, P.L. Functionalized de novo designed proteins: mechanism of proton coupling to oxidation/reduction in heme protein maquettes. Biochemistry 37, 16815–16827 (1998).

    Article  CAS  Google Scholar 

  12. Grosset, A.M., Gibney, B.R., Rabanal, F., Moser, C.C. & Dutton, P.L. Proof of principle in a de novo designed protein maquette: an allosterically regulated, charge-activated conformational switch in a tetra-α-helix bundle. Biochemistry 40, 5474–5487 (2001).

    Article  CAS  Google Scholar 

  13. Anderson, J.L.R., Koder, R.L., Moser, C.C. & Dutton, P.L. Controlling complexity and water penetration in functional de novo protein design. Biochem. Soc. Trans. 36, 1106–1111 (2008).

    Article  CAS  Google Scholar 

  14. Koder, R.L. et al. Design and engineering of an O2 transport protein. Nature 458, 305–309 (2009).

    Article  CAS  Google Scholar 

  15. Bender, G.M. et al. De novo design of a single-chain diphenylporphyrin metalloprotein. J. Am. Chem. Soc. 129, 10732–10740 (2007).

    Article  CAS  Google Scholar 

  16. Ku, J. & Schultz, P.G. Alternate protein frameworks for molecular recognition. Proc. Natl. Acad. Sci. USA 92, 6552–6556 (1995).

    Article  CAS  Google Scholar 

  17. Predki, P.F. & Regan, L. Redesigning the topology of a four-helix-bundle protein: monomeric Rop. Biochemistry 34, 9834–9839 (1995).

    Article  CAS  Google Scholar 

  18. Westerlund, K. et al. Making a single-chain four-helix bundle for redox chemistry studies. Protein Eng. Des. Sel. 21, 645–652 (2008).

    Article  CAS  Google Scholar 

  19. Huang, S.S., Gibney, B.R., Stayrook, S.E., Dutton, P.L. & Lewis, M. X-ray structure of a Maquette scaffold. J. Mol. Biol. 326, 1219–1225 (2003).

    Article  CAS  Google Scholar 

  20. Aurora, R. & Rose, G.D. Helix capping. Protein Sci. 7, 21–38 (1998).

    Article  CAS  Google Scholar 

  21. Koder, R.L. et al. Native like structure in designed four α-helix bundles driven by buried polar interactions. J. Am. Chem. Soc. 128, 14450–14451 (2006).

    Article  CAS  Google Scholar 

  22. Caffrey, M.S. & Cusanovich, M.A. Site-specific mutagenesis studies of cytochromes c. Biochim. Biophys. Acta 1187, 277–288 (1994).

    Article  CAS  Google Scholar 

  23. Davies, A.M. et al. Redesign of the interior hydrophilic region of mitochondrial cytochrome c by site-directed mutagenesis. Biochemistry 32, 5431–5435 (1993).

    Article  CAS  Google Scholar 

  24. Trent, J.T., Hvitved, A.N. & Hargrove, M.S. A model for ligand binding to hexacoordinate hemoglobins. Biochemistry 40, 6155–6163 (2001).

    Article  CAS  Google Scholar 

  25. Kiger, L. et al. Electron transfer function versus oxygen delivery: a comparative study for several hexacoordinated globins across the animal kingdom. PLoS ONE 6, e20478 (2011).

    Article  CAS  Google Scholar 

  26. Hamdane, D. et al. Hyperthermal stability of neuroglobin and cytoglobin. FEBS J. 272, 2076–2084 (2005).

    Article  CAS  Google Scholar 

  27. Fago, A., Mathews, A.J., Moens, L., Dewilde, S. & Brittain, T. The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c. FEBS Lett. 580, 4884–4888 (2006).

    Article  CAS  Google Scholar 

  28. Margoliash, E. & Bosshard, H.R. Guided by electrostatics, a textbook protein comes of age. Trends Biochem. Sci. 8, 316–320 (1983).

    Article  CAS  Google Scholar 

  29. Moser, C.C. & Dutton, P.L. Cytochrome c and c2 binding dynamics and electron transfer with photosynthetic reaction center protein and other integral membrane redox proteins. Biochemistry 27, 2450–2461 (1988).

    Article  CAS  Google Scholar 

  30. Ku, H.H., Brunk, U.T. & Sohal, R.S. Relationship between mitochondrial superoxide and hydrogen-peroxide production and longevity of mammalian-species. Free Radic. Biol. Med. 15, 621–627 (1993).

    Article  CAS  Google Scholar 

  31. Muir Wood, P. The redox potential of the system oxygen—superoxide. FEBS Lett. 44, 22–24 (1974).

    Article  CAS  Google Scholar 

  32. Zhang, L., Andersen, E.M.E., Khajo, A., Magliozzo, R.S. & Koder, R.L. Dynamic factors affecting gaseous ligand binding in an artificial oxygen transport protein. Biochemistry 52, 447–455 (2013).

    Article  CAS  Google Scholar 

  33. Cross, A.R., Parkinson, J.F. & Jones, O.T.G. Mechanism of the superoxide-producing oxidase of neutrophils—O2 is necessary for the fast reduction of cytochrome b245 by NADPH. Biochem. J. 226, 881–884 (1985).

    Article  CAS  Google Scholar 

  34. Lin, C. et al. Association of flavin adenine-dinucleotide with the Arabidopsis blue-light receptor Cry1. Science 269, 968–970 (1995).

    Article  CAS  Google Scholar 

  35. Aubert, C., Vos, M.H., Mathis, P., Eker, A.P. & Brettel, K. Intraprotein radical transfer during photoactivation of DNA photolyase. Nature 405, 586–590 (2000).

    Article  CAS  Google Scholar 

  36. Muthiah, C., Ptaszek, M., Nguyen, T.M., Flack, K.M. & Lindsey, J.S. Two complementary routes to 7-substituted chlorins. Partial mimics of chlorophyll b. J. Org. Chem. 72, 7736–7749 (2007).

    Article  CAS  Google Scholar 

  37. Moser, C.C., Anderson, J.L. & Dutton, P.L. Guidelines for tunneling in enzymes. Biochim. Biophys. Acta 1797, 1573–1586 (2010).

    Article  CAS  Google Scholar 

  38. Moser, C.C., Keske, J.M., Warncke, K., Farid, R.S. & Dutton, P.L. Nature of biological electron transfer. Nature 355, 796–802 (1992).

    Article  CAS  Google Scholar 

  39. Massey, V., Stankovich, M. & Hemmerich, P. Light-mediated reduction of flavoproteins with flavins as catalysts. Biochemistry 17, 1–8 (1978).

    Article  CAS  Google Scholar 

  40. Grzyb, J. et al. Empirical and computational design of iron-sulfur cluster proteins. Biochim. Biophys. Acta 1817, 1256–1262 (2012).

    Article  CAS  Google Scholar 

  41. Reedy, C.J. & Gibney, B.R. Heme protein assemblies. Chem. Rev. 104, 617–649 (2004).

    Article  CAS  Google Scholar 

  42. Monien, B.H., Drepper, F., Sommerhalter, M., Lubitz, W. & Haehnel, W. Detection of heme oxygenase activity in a library of four-helix bundle proteins: towards the de novo synthesis of functional heme proteins. J. Mol. Biol. 371, 739–753 (2007).

    Article  CAS  Google Scholar 

  43. Patel, S.C. & Hecht, M.H. Directed evolution of the peroxidase activity of a de novo–designed protein. Protein Eng. Des. Sel. 25, 445–452 (2012).

    Article  CAS  Google Scholar 

  44. Smith, B.A. & Hecht, M.H. Novel proteins: from fold to function. Curr. Opin. Chem. Biol. 15, 421–426 (2011).

    Article  CAS  Google Scholar 

  45. Fry, H.C., Lehmann, A., Saven, J.G., DeGrado, W.F. & Therien, M.J. Computational design and elaboration of a de novo heterotetrameric α-helical protein that selectively binds an emissive abiological (porphinato)zinc chromophore. J. Am. Chem. Soc. 132, 3997–4005 (2010).

    Article  CAS  Google Scholar 

  46. Reig, A.J. et al. Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins. Nat. Chem. 4, 900–906 (2012).

    Article  CAS  Google Scholar 

  47. Röthlisberger, D. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195 (2008).

    Article  Google Scholar 

  48. Miner, K.D. et al. A designed functional metalloenzyme that reduces O2 to H2O with over one thousand turnovers. Angew. Chem. Int. Ed. Engl. 51, 5589–5592 (2012).

    Article  CAS  Google Scholar 

  49. Gibney, B.R., Mulholland, S.E., Rabanal, F. & Dutton, P.L. Ferredoxin and ferredoxin-heme maquettes. Proc. Natl. Acad. Sci. USA 93, 15041–15046 (1996).

    Article  CAS  Google Scholar 

  50. Fisher, M.A., McKinley, K.L., Bradley, L.H., Viola, S.R. & Hecht, M.H. De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth. PLoS ONE 6, e15364 (2011).

    Article  CAS  Google Scholar 

  51. Berry, E.A. & Trumpower, B.L. Simultaneous determination of hemes a, b and c from pyridine hemochrome spectra. Anal. Biochem. 161, 1–15 (1987).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

In this research, the US National Institutes of Health (NIH)–General Medical Institutes (RO1 GM 41048) funded the design and development of the maquette proteins A, B, F, G and H, including gene design, cloning, protein expression, purification and characterization; it also funded the thermal stability measurements using CD and demonstrations of control of oxygen binding and redox chemistry. Basic to these developments was NMR spectroscopy performed by M.A.S., K.G.V. and A.J.W., supported by NIH United States Public Health Service grants DK39806 and GM102477 to A.J.W. The US Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering (DE-FG02-05ER46223) funded the synthesis and characterization of flavins and also the design, expression, purification and characterization of C, K, J protein maquettes promoting light-activated charge separation and oxidation-reduction using flavin and Zn- and Fe- tetrapyrroles as cofactors. The US Department of Energy Office of Basic Energy Sciences, Energy Frontier Research Center (PARC) (DE-SC 0001035 to P.L.D. and C.C.M.) funded development of light excitation energy transfer in maquettes (L and its mutants), synthesis and purification of Zn pyropheophoribide a and covalent attachments of Alexa Fluor to the maquettes. In this work, the synthetic chlorin ZnC was a generous gift from O. Mass and J.S. Lindsey at North Carolina State University.

Author information

Author notes

  1. Tammer A Farid, Goutham Kodali and Lee A Solomon: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Tammer A Farid, Goutham Kodali, Lee A Solomon, Bruce R Lichtenstein, Molly M Sheehan, Bryan A Fry, Chris Bialas, Nathan M Ennist, Jessica A Siedlecki, Zhenyu Zhao, Matthew A Stetz, Kathleen G Valentine, J L Ross Anderson, A Joshua Wand, Bohdana M Discher, Christopher C Moser & P Leslie Dutton

  2. Max Planck Institute for Developmental Biology, Tübingen, Germany

    Bruce R Lichtenstein

  3. School of Biochemistry, University of Bristol, Bristol, UK

    J L Ross Anderson

Authors
  1. Tammer A Farid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Goutham Kodali
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lee A Solomon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bruce R Lichtenstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Molly M Sheehan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bryan A Fry
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chris Bialas
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Nathan M Ennist
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jessica A Siedlecki
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zhenyu Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Matthew A Stetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Kathleen G Valentine
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. J L Ross Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. A Joshua Wand
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Bohdana M Discher
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Christopher C Moser
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. P Leslie Dutton
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.A.F. contributed to the design and characterization of maquettes A, E, J, H and B; G.K. designed, expressed, purified and characterized maquettes C, L, D, K and F and contributed to all of the experiments performed with these maquettes as well as the writing of the manuscript; L.A.S. performed the redox titrations, developed and characterized maquette G and performed CO and O2 kinetics on maquette A as well as assisted in the writing of the manuscript; B.R.L. contributed to the monomeric maquette design as well as experimental design and interpretation; M.M.S. designed and purified maquette I and measured superoxide production, low-temperature spectra and oxyferrous state kinetics; B.A.F. measured electron transfer from A to cytochrome c; C.B. synthesized and characterized flavomaquettes; N.M.E. performed synthetic chlorin binding affinity measurements and contributed to protein design; J.A.S. contributed to heme and Zn porphyrin binding affinities of C and J; Z.Z. contributed to protein design; B.M.D. contributed to experimental designs and manuscript writing; M.A.S., K.G.V. and A.J.W. contributed to the NMR characterization of maquettes; J.L.R.A. contributed to CO and O2 ligand kinetics for maquette A. C.C.M. designed and operated transient spectroscopy equipment for photolysis and light-induced electron transfer and contributed substantially to manuscript writing; P.L.D. conceived and designed experiments and contributed substantially to manuscript writing.

Corresponding author

Correspondence to P Leslie Dutton.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–43. (PDF 4867 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Farid, T., Kodali, G., Solomon, L. et al. Elementary tetrahelical protein design for diverse oxidoreductase functions. Nat Chem Biol 9, 826–833 (2013). https://doi.org/10.1038/nchembio.1362

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1362

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing