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Design of functional metalloproteins

Nature volume 460, pages 855–862 (2009)Cite this article

Abstract

Metalloproteins catalyse some of the most complex and important processes in nature, such as photosynthesis and water oxidation. An ultimate test of our knowledge of how metalloproteins work is to design new metalloproteins. Doing so not only can reveal hidden structural features that may be missing from studies of native metalloproteins and their variants, but also can result in new metalloenzymes for biotechnological and pharmaceutical applications. Although it is much more challenging to design metalloproteins than non-metalloproteins, much progress has been made in this area, particularly in functional design, owing to recent advances in areas such as computational and structural biology.

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Figure 1: Designed metalloproteins using de novo-designed scaffolds.
Figure 2: Designed metalloproteins using native scaffolds.
Figure 3: Site-specific incorporation of unnatural amino acids into a protein scaffold for the tuning of metalloprotein functional properties.
Figure 4: Examples of strategies for the incorporation of non-native metal cofactors into protein scaffolds.
Figure 5: Close match between a designed metalloprotein and its native target protein.

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References

  1. Lu, Y., Berry, S. M. & Pfister, T. D. Engineering novel metalloproteins: design of metal-binding sites into native protein scaffolds. Chem. Rev. 101, 3047–3080 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Barker, P. D. Designing redox metalloproteins from bottom-up and top-down perspectives. Curr. Opin. Struct. Biol. 13, 490–499 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Lu, Y. Metalloprotein and metallo-DNA/RNAzyme design: current approaches, success measures and future challenges. Inorg. Chem. 45, 9930–9940 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. DeGrado, W. F., Summa, C. M., Pavone, V., Nastri, F. & Lombardi, A. De novo design and structural characterization of proteins and metalloproteins. Annu. Rev. Biochem. 68, 779–819 (1999).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Choma, C. T. et al. Design of a heme-binding four-helix bundle. J. Am. Chem. Soc. 116, 856–865 (1994).

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  8. Case, M. A. & McLendon, G. L. Metal-assembled modular proteins: toward functional protein design. Acc. Chem. Res. 37, 754–762 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Huang, S. S., Koder, R. L., Lewis, M., Wand, A. J. & Dutton, P. L. The HP-1 maquette: from an apoprotein structure to a structured hemoprotein designed to promote redox-coupled proton exchange. Proc. Natl Acad. Sci. USA 101, 5536–5541 (2004).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Sasaki, T. & Kaiser, E. T. Helichrome: synthesis and enzymic activity of a designed hemeprotein. J. Am. Chem. Soc. 111, 380–381 (1989).

    Article  CAS  Google Scholar 

  11. Das, A. & Hecht, M. H. Peroxidase activity of de novo heme proteins immobilized on electrodes. J. Inorg. Biochem. 101, 1820–1826 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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  PubMed  Google Scholar 

  13. Koder, R. L. et al. Design and engineering of an O2 transport protein. Nature 458, 305–309 (2009). This paper reports the design of an O 2 transport protein that has O 2 affinities and exchange timescales that match those of natural globins.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Klemba, M. & Regan, L. Characterization of metal binding by a designed protein: single ligand substitutions at a tetrahedral Cys2His2 site. Biochemistry 34, 10094–10100 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. Handel, T. & DeGrado, W. F. De novo design of a Zn2+-binding protein. J. Am. Chem. Soc. 112, 6710–6711 (1990).

    Article  CAS  Google Scholar 

  16. Touw, D. S., Nordman, C. E., Stuckey, J. A. & Pecoraro, V. L. Identifying important structural characteristics of arsenic resistance proteins by using designed three-stranded coiled coils. Proc. Natl Acad. Sci. USA 104, 11969–11974 (2007).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lombardi, A. et al. Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins. Proc. Natl Acad. Sci. USA 97, 6298–6305 (2000). This paper reports the use of a novel method to design functional metalloproteins that do not contain haem.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kaplan, J. & DeGrado, W. F. De novo design of catalytic proteins. Proc. Natl Acad. Sci. USA 101, 11566–11570 (2004).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Calhoun, J. R. et al. Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the 'intermediate' and reaction mechanism. J. Am. Chem. Soc. 130, 9188–9189 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Matzapetakis, M. et al. Comparison of the binding of cadmium(II), mercury(II), and arsenic(III) to the de novo designed peptides TRI L12C and TRI L16C. J. Am. Chem. Soc. 124, 8042–8054 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Ghosh, D., Lee, K. H., Demeler, B. & Pecoraro, V. L. Linear free-energy analysis of mercury(II) and cadmium(II) binding to three-stranded coiled coils. Biochemistry 44, 10732–10740 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Petros, A. K., Reddi, A. R., Kennedy, M. L., Hyslop, A. G. & Gibney, B. R. Femtomolar Zn(II) affinity in a peptide-based ligand designed to model thiolate-rich metalloprotein active sites. Inorg. Chem. 45, 9941–9958 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Dieckmann, G. R. et al. De novo design of mercury-binding two- and three-helical bundles. J. Am. Chem. Soc. 119, 6195–6196 (1997).

    Article  CAS  Google Scholar 

  24. Ghosh, D. & Pecoraro, V. L. Probing metal–protein interactions using a de novo design approach. Curr. Opin. Chem. Biol. 9, 97–103 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Kharenko, O. A. & Ogawa, M. Y. Metal-induced folding of a designed metalloprotein. J. Inorg. Biochem. 98, 1971–1974 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Farrer, B. T. & Pecoraro, V. L. Hg(II) binding to a weakly associated coiled coil nucleates an encoded metalloprotein fold: a kinetic analysis. Proc. Natl Acad. Sci. USA 100, 3760–3765 (2003).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Iranzo, O., Jakusch, T., Lee, K.-H., Hemmingsen, L. & Pecoraro, V. L. The correlation of 113Cd NMR and 111mCd PAC spectroscopies provides a powerful approach for the characterization of the structure of Cd(II)-substituted Zn(II) proteins. Chem. Eur. J. 15, 3761–3772 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Peacock, A. F. A., Iranzo, O. & Pecoraro, V. L. Harnessing nature's ability to control metal ion coordination geometry using de novo designed peptides. Dalton Trans. 2271–2280 (2009). This paper describes an excellent demonstration of the use of a de novo -designed peptide to control metal-binding-site geometry.

  29. Nanda, V. et al. De novo design of a redox-active minimal rubredoxin mimic. J. Am. Chem. Soc. 127, 5804–5805 (2005). This paper reports a rare example of a designed metalloprotein in a de novo -designed β -structure-containing protein.

    Article  CAS  PubMed  Google Scholar 

  30. Kono, H. & Saven, J. G. Statistical theory for protein combinatorial libraries. Packing interactions, backbone flexibility, and the sequence variability of a main-chain structure. J. Mol. Biol. 306, 607–628 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Williams, R. J. P. Energised (entatic) states of groups and of secondary structures in proteins and metalloproteins. Eur. J. Biochem. 234, 363–381 (1995).

    Article  CAS  PubMed  Google Scholar 

  32. Ueno, T., Ohki, T. & Watanabe, Y. Molecular engineering of cytochrome P 450 and myoglobin for selective oxygenations. J. Porphyrins Phthalocyanines 8, 279–289 (2004).

    Article  CAS  Google Scholar 

  33. Yeung, N. & Lu, Y. One heme, diverse functions: using biosynthetic myoglobin models to gain insights into heme copper oxidases and nitric oxide reductases. Chem. Biodivers. 5, 1437–1454 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ozaki, S.-I., Matsui, T. & Watanabe, Y. Conversion of myoglobin into a highly stereospecific peroxygenase by the L29H/H64L mutation. J. Am. Chem. Soc. 118, 9784–9785 (1996).

    Article  CAS  Google Scholar 

  35. Jensen, K. K., Martini, L. & Schwartz, T. W. Enhanced fluorescence resonance energy transfer between spectral variants of green fluorescent protein through zinc-site engineering. Biochemistry 40, 938–945 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Evers, T. H., Appelhof, M. A., de Graaf-Heuvelmans, P. T., Meijer, E. W. & Merkx, M. Ratiometric detection of Zn(II) using chelating fluorescent protein chimeras. J. Mol. Biol. 374, 411–425 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Mizuno, T., Murao, K., Tanabe, Y., Oda, M. & Tanaka, T. Metal-ion dependent GFP emission in vivo by combining a circularly permutated green fluorescent protein with an engineered metal ion-binding coiled-coil. J. Am. Chem. Soc. 129, 11378–11383 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Wegner, S. V., Boyaci, H., Chen, H., Jensen, M. P. & He, C. Engineering a uranyl-specific binding protein from NikR. Angew. Chem. Int. Edn Engl. 48, 2339–2341 (2009).

    Article  CAS  Google Scholar 

  39. Salgado, E. N., Faraone-Mennella, J. & Tezcan, F. A. Controlling protein–protein interactions through metal coordination: assembly of a 16-helix bundle protein. J. Am. Chem. Soc. 129, 13374–13375 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Matthews, J. M., Loughlin, F. E. & Mackay, J. P. Designed metal-binding sites in biomolecular and bioinorganic interactions. Curr. Opin. Struct. Biol. 18, 484–490 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Park, H. S. et al. Design and evolution of new catalytic activity with an existing protein scaffold. Science 311, 535–538 (2006).

    Article  ADS  CAS  PubMed  Google Scholar 

  42. Vita, C., Roumestand, C., Tom, F. & Menez, A. Scorpion toxins as natural scaffolds for protein engineering. Proc. Natl Acad. Sci. USA 92, 6404–6408 (1995).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  43. Müller, H. N. & Skerra, A. Grafting of a high-affinity Zn(II)-binding site on the β-barrel of retinol-binding protein results in enhanced folding stability and enables simplified purification. Biochemistry 33, 14126–14135 (1994).

    Article  PubMed  Google Scholar 

  44. Desjarlais, J. R. & Clarke, N. D. Computer search algorithms in protein modification and design. Curr. Opin. Struct. Biol. 8, 471–475 (1998).

    Article  CAS  PubMed  Google Scholar 

  45. Benson, D. E., Wisz, M. S., Liu, W. & Hellinga, H. W. Construction of a novel redox protein by rational design: conversion of a disulfide bridge into a mononuclear iron–sulfur center. Biochemistry 37, 7070–7076 (1998).

    Article  CAS  PubMed  Google Scholar 

  46. Yang, W. et al. Rational design of a calcium-binding protein. J. Am. Chem. Soc. 125, 6165–6171 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Shete, V. S. & Benson, D. E. Protein design provides lead(II) ion biosensors for imaging molecular fluxes around red blood cells. Biochemistry 48, 462–470 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Hay, M., Richards, J. H. & Lu, Y. Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase. Proc. Natl Acad. Sci. USA 93, 461–464 (1996).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  49. Robinson, H. et al. Structural basis of electron transfer modulation in the purple CuA center. Biochemistry 38, 5677–5683 (1999).

    Article  CAS  PubMed  Google Scholar 

  50. Dennison, C., Vijgenboom, E., de Vries, S., van der Oost, J. & Canters, G. W. Introduction of a CuA site into the blue copper protein amicyanin from Thiobacillus versutus . FEBS Lett. 365, 92–94 (1995).

    Article  CAS  PubMed  Google Scholar 

  51. Jones, L. H., Liu, A. & Davidson, V. L. An engineered CuA amicyanin capable of intermolecular electron transfer reactions. J. Biol. Chem. 278, 47269–47274 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Franklin, S. J. & Welch, J. T. The helix-turn-helix as a scaffold for chimeric nuclease design. Comments Inorg. Chem. 26, 127–164 (2005).

    Article  CAS  Google Scholar 

  53. Sigman, J. A., Kwok, B. C. & Lu, Y. From myoglobin to heme–copper oxidase: design and engineering of a CuB center into sperm whale myoglobin. J. Am. Chem. Soc. 122, 8192–8196 (2000).

    Article  CAS  Google Scholar 

  54. Sigman, J. A., Kim, H. K., Zhao, X., Carey, J. R. & Lu, Y. The role of copper and protons in heme–copper oxidases: kinetic study of an engineered heme–copper center in myoglobin. Proc. Natl Acad. Sci. USA 100, 3629–3634 (2003). This paper presents a good illustration of the role of secondary coordination spheres, such as proton networks, in metalloprotein design.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  55. Zhao, X., Yeung, N., Russell, B. S., Garner, D. K. & Lu, Y. Catalytic reduction of NO to N2O by a designed heme copper center in myoglobin: implications for the role of metal ions. J. Am. Chem. Soc. 128, 6766–6767 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Yeung, B. K. S., Wang, X., Sigman, J. A., Petillo, P. A. & Lu, Y. Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase. Chem. Biol. 4, 215–221 (1997).

    Article  CAS  PubMed  Google Scholar 

  57. Abe, S., Ueno, T. & Watanabe, Y. Artificial metalloproteins exploiting vacant space: preparation, structures, and functions. Top. Organomet. Chem. 25, 25–43 (2009).

    Article  CAS  Google Scholar 

  58. Ueno, T. et al. Size-selective olefin hydrogenation by a Pd nanocluster provided in an apo-ferritin cage. Angew. Chem. Int. Edn Engl. 43, 2527–2530 (2004).

    Article  CAS  Google Scholar 

  59. Varpness, Z., Peters, J. W., Young, M. & Douglas, T. Biomimetic synthesis of a H2 catalyst using a protein cage architecture. Nano Lett. 5, 2306–2309 (2005).

    Article  ADS  CAS  PubMed  Google Scholar 

  60. Yin, J., Mills, J. H. & Schultz, P. G. A catalysis-based selection for peroxidase antibodies with increased activity. J. Am. Chem. Soc. 126, 3006–3007 (2004).

    Article  CAS  PubMed  Google Scholar 

  61. Rasmussen, B. S. et al. Enantioselective proteins: selection, binding studies and molecular modeling of antibodies with affinity towards hydrophobic BINOL derivatives. Chembiochem 8, 1974–1980 (2007).

    Article  CAS  PubMed  Google Scholar 

  62. Fasan, R., Chen, M. M., Crook, N. C. & Arnold, F. H. Engineered alkane-hydroxylating cytochrome P450BM3 exhibiting nativelike catalytic properties. Angew. Chem. Int. Edn Engl. 46, 8414–8418 (2007). This paper describes a good example of evolving metalloproteins with new and more demanding activities.

    Article  CAS  Google Scholar 

  63. Lu, Y. Design and engineering of metalloproteins containing unnatural amino acids or non-native metal-containing cofactors. Curr. Opin. Chem. Biol. 9, 118–126 (2005). This paper is a good review of the emerging field of metalloprotein design using unnatural amino acids and non-native metal-containing cofactors.

    Article  CAS  PubMed  Google Scholar 

  64. Merrifield, B. Concept and early development of solid-phase peptide synthesis. Methods Enzymol. 289, 3–13 (1997).

    Article  CAS  PubMed  Google Scholar 

  65. Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. H. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  66. Muir, T. W. Semisynthesis of proteins by expressed protein ligation. Annu. Rev. Biochem. 72, 249–289 (2003).

    Article  CAS  PubMed  Google Scholar 

  67. Ikeda, Y. et al. Synthesis of a novel histidine analog and its efficient incorporation into a protein in vivo . Protein Eng. 16, 699–706 (2003).

    Article  CAS  PubMed  Google Scholar 

  68. Qi, D., Tann, C.-M., Haring, D. & Distefano, M. D. Generation of new enzymes via covalent modification of existing proteins. Chem. Rev. 101, 3081–3111 (2001).

    Article  CAS  PubMed  Google Scholar 

  69. Barrick, D. Depletion and replacement of protein metal ligands. Curr. Opin. Biotechnol. 6, 411–418 (1995).

    Article  CAS  PubMed  Google Scholar 

  70. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C. & Schultz, P. G. A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–188 (1989).

    Article  ADS  CAS  PubMed  Google Scholar 

  71. Lee, H. S. & Schultz, P. G. Biosynthesis of a site-specific DNA cleaving protein. J. Am. Chem. Soc. 130, 13194–13195 (2008). This paper reports the first example of introducing unnatural amino acids that bind metal ions with functional properties.

    Article  CAS  PubMed  Google Scholar 

  72. Privett, H. K., Reedy, C. J., Kennedy, M. L. & Gibney, B. R. Nonnatural amino acid ligands in heme protein design. J. Am. Chem. Soc. 124, 6828–6829 (2002).

    Article  CAS  PubMed  Google Scholar 

  73. Petros, A. K., Shaner, S. E., Costello, A. L., Tierney, D. L. & Gibney, B. R. Comparison of cysteine and penicillamine ligands in a Co(II) maquette. Inorg. Chem. 43, 4793–4795 (2004).

    Article  CAS  PubMed  Google Scholar 

  74. Lee, K.-H., Cabello, C., Hemmingsen, L., Marsh, E. N. G. & Pecoraro, V. L. Using nonnatural amino acids to control metal-coordination number in three-stranded coiled coils. Angew. Chem. Int. Edn Engl. 45, 2864–2868 (2006).

    Article  CAS  Google Scholar 

  75. Peacock, A. F. A., Hemmingsen, L. & Pecoraro, V. L. Using diastereopeptides to control metal ion coordination in proteins. Proc. Natl Acad. Sci. USA 105, 16566–16571 (2008).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  76. Low, D. W. & Hill, M. G. Rational fine-tuning of the redox potentials in chemically synthesized rubredoxins. J. Am. Chem. Soc. 120, 11536–11537 (1998).

    Article  CAS  Google Scholar 

  77. Low, D. W. & Hill, M. G. Backbone-engineered high-potential iron proteins: effects of active-site hydrogen binding on reduction potential. J. Am. Chem. Soc. 122, 11039–11040 (2000). This paper demonstrates the power of using unnatural amino acids in tuning metalloprotein redox potentials using backbone positions.

    Article  CAS  Google Scholar 

  78. Berry, S. M., Gieselman, M. D., Nilges, M. J., Van der Donk, W. A. & Lu, Y. An engineered azurin variant containing a selenocysteine copper ligand. J. Am. Chem. Soc. 124, 2084–2085 (2002).

    Article  CAS  PubMed  Google Scholar 

  79. Berry, S. M., Ralle, M., Low, D. W., Blackburn, N. J. & Lu, Y. Probing the role of axial methionine in the blue copper center of azurin with unnatural amino acids. J. Am. Chem. Soc. 125, 8760–8768 (2003). This paper presents a clear demonstration of using isostructural unnatural amino acids in fine-tuning the redox properties of metalloproteins.

    Article  CAS  PubMed  Google Scholar 

  80. Hayashi, T. & Hisaeda, Y. New functionalization of myoglobin by chemical modification of heme-propionates. Acc. Chem. Res. 35, 35–43 (2002).

    Article  CAS  PubMed  Google Scholar 

  81. Matsuo, T., Hayashi, T. & Hisaeda, Y. Reductive activation of dioxygen by a myoglobin reconstituted with a flavohemin. J. Am. Chem. Soc. 124, 11234–11235 (2002).

    Article  CAS  PubMed  Google Scholar 

  82. Hayashi, T. et al. Crystal structure and peroxidase activity of myoglobin reconstituted with iron porphycene. Inorg. Chem. 45, 10530–10536 (2006).

    Article  CAS  PubMed  Google Scholar 

  83. Cochran, F. V. et al. Computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor. J. Am. Chem. Soc. 127, 1346–1347 (2005).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Ohashi, M. et al. Preparation of artificial metalloenzymes by insertion of chromium Schiff base complexes into apomyoglobin mutants. Angew. Chem. Int. Edn Engl. 42, 1005–1008 (2003).

    Article  CAS  Google Scholar 

  86. Ueno, T. et al. Crystal structures of artificial metalloproteins: tight binding of FeIII(Schiff-base) by mutation of Ala71 to Gly in apo-myoglobin. Inorg. Chem. 43, 2852–2858 (2004).

    Article  CAS  PubMed  Google Scholar 

  87. Wilson, M. E. & Whitesides, G. M. Conversion of a protein to a homogeneous asymmetric hydrogenation catalyst by site-specific modification with a diphosphinerhodium(I) moiety. J. Am. Chem. Soc. 100, 306–307 (1978).

    Article  CAS  Google Scholar 

  88. Collot, J. et al. Artificial metalloenzymes for enantioselective catalysis based on biotin–avidin. J. Am. Chem. Soc. 125, 9030–9031 (2003).

    Article  CAS  PubMed  Google Scholar 

  89. Steinreiber, J. & Ward, T. R. Artificial metalloenzymes as selective catalysts in aqueous media. Coord. Chem. Rev. 252, 751–766 (2008).

    Article  CAS  Google Scholar 

  90. Letondor, C. et al. Artificial transfer hydrogenases based on the biotin–(strept)avidin technology: fine tuning the selectivity by saturation mutagenesis of the host protein. J. Am. Chem. Soc. 128, 8320–8328 (2006).

    Article  CAS  PubMed  Google Scholar 

  91. Creus, M. et al. X-ray structure and designed evolution of an artificial transfer hydrogenase. Angew. Chem. Int. Edn Engl. 47, 1400–1404 (2008).

    Article  CAS  Google Scholar 

  92. Chen, C.-H. B., Milne, L., Landgraf, R., Perrin, D. M. & Sigman, D. S. Artificial nucleases. Chembiochem 2, 735–740 (2001).

    Article  CAS  PubMed  Google Scholar 

  93. Davies, R. R. & Distefano, M. D. A semisynthetic metalloenzyme based on a protein cavity that catalyzes the enantioselective hydrolysis of ester and amide substrates. J. Am. Chem. Soc. 119, 11643–11652 (1997).

    Article  CAS  Google Scholar 

  94. Ory, J. J. et al. Structural characterization of two synthetic catalysts based on adipocyte lipid-binding protein. Protein Eng. 11, 253–261 (1998).

    Article  CAS  PubMed  Google Scholar 

  95. Carey, J. R. et al. A site-selective dual anchoring strategy for artificial metalloprotein design. J. Am. Chem. Soc. 126, 10812–10813 (2004).

    Article  CAS  PubMed  Google Scholar 

  96. Zhang, J., Garner, D. K., Liang, L., Chen, Q. & Lu, Y. Protein scaffold of a designed metalloenzyme enhances the chemoselectivity in sulfoxidation of thioanisole. Chem. Commun. 1665–1667 (2008).

  97. Farver, O., Lu, Y., Ang, M. C. & Pecht, I. Enhanced rate of intramolecular electron transfer in an engineered purple CuA azurin. Proc. Natl Acad. Sci. USA 96, 899–902 (1999). This paper presents an excellent demonstration of the power of metalloprotein design to place two metal-binding sites into the same protein scaffold for direct comparison of functional properties.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  98. Wang, N., Zhao, X. & Lu, Y. Role of heme types in heme–copper oxidases: effects of replacing a heme b with a heme o mimic in an engineered heme–copper center in myoglobin. J. Am. Chem. Soc. 127, 16541–16547 (2005).

    Article  CAS  PubMed  Google Scholar 

  99. Summa, C. M., Rosenblatt, M. M., Hong, J.-K., Lear, J. D. & DeGrado, W. F. Computational de novo design, and characterization of an A2B2 diiron protein. J. Mol. Biol. 321, 923–938 (2002).

    Article  CAS  PubMed  Google Scholar 

  100. Bloom, J. D., Labthavikul, S. T., Otey, C. R. & Arnold, F. H. Protein stability promotes evolvability. Proc. Natl Acad. Sci. USA 103, 5869–5874 (2006).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank W. F. DeGrado, B. R. Gibney and P. L. Dutton for providing images used in Figure 1, N. Nagraj for help with editing the manuscript, and the US National Science Foundation (CHE 05-52008) and National Institutes of Health (GM062211) for financial support.

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  1. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Yi Lu, Natasha Yeung, Nathan Sieracki & Nicholas M. Marshall

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Lu, Y., Yeung, N., Sieracki, N. et al. Design of functional metalloproteins. Nature 460, 855–862 (2009). https://doi.org/10.1038/nature08304

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