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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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.
The authors declare no competing financial interests.
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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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