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Elementary tetrahelical protein design for diverse oxidoreductase functions

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

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A Corrigendum to this article was published on 17 January 2014

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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.

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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.

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  • 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.

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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.

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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

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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.

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Correspondence to P Leslie Dutton.

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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

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