Article | Published:

A protein engineered to bind uranyl selectively and with femtomolar affinity

Nature Chemistry volume 6, pages 236241 (2014) | Download Citation

Abstract

Uranyl (UO22+), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 109 (13.7 nM); however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30–60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    A fresh look at element distribution in the North Pacific. Eos Trans. Am. Geophys. Union 78, 221–227 (1997).

  2. 2.

    , & Extraction of uranium from seawater by polymer-bound macrocyclic hexaketone. Nature 280, 665–666 (1979).

  3. 3.

    Present status of study on extraction of uranium from sea water. J. Nucl. Sci. Technol. 21, 1–9 (1984).

  4. 4.

    , & Recovery of uranium from seawater. 7. Concentration and separation of uranium in acidic eluate. Ind. Eng. Chem. Res. 29, 2273–2277 (1990).

  5. 5.

    , , & Selective recognition and extraction of the uranyl ion. J. Am. Chem. Soc. 132, 13572–13574 (2010).

  6. 6.

    , & Stereognostic coordination chemistry. 1. The design and synthesis of chelators for the uranyl ion. J. Am. Chem. Soc. 114, 8138–8146 (1992).

  7. 7.

    , , & Rational design of sequestering agents for plutonium and other actinides. Chem. Rev. 103, 4207–4282 (2003).

  8. 8.

    , , , & A ‘Texas-sized’ molecular box that forms an anion-induced supramolecular necklace. Nature Chem. 2, 406–409 (2010).

  9. 9.

    , , , & Engineering a uranyl-specific binding protein from NikR. Angew. Chem. Int. Ed. 48, 2339–2341 (2009).

  10. 10.

    , , & Highly sensitive and selective colorimetric sensors for uranyl (UO22+): development and comparison of labeled and label-free DNAzyme–gold nanoparticle systems. J. Am. Chem. Soc. 130, 14217–14226 (2008).

  11. 11.

    & Selective binding of uranyl cation by a novel calmodulin peptide. Environ. Chem. Lett. 4, 45–49 (2006).

  12. 12.

    & Layered metal sulfides capture uranium from seawater. J. Am. Chem. Soc., 134 (39), 16441–16446 (2012).

  13. 13.

    , , & Highly porous and stable metal–organic frameworks for uranium extraction. Chem. Sci. 4, 2396–2402 (2013).

  14. 14.

    et al. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383–1387 (2003).

  15. 15.

    , & Stoichiometric and site characteristics of the binding of iron to human transferrin. J. Biol. Chem. 253, 1930–1937 (1978).

  16. 16.

    et al. Affinity gradients drive copper to cellular destinations. Nature 465, 367–368 (2010).

  17. 17.

    et al. Display of beta-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp′–OmpA′–beta-lactamase fusions. Protein Eng. 9, 239–247 (1996).

  18. 18.

    & Automatch: target-binding protein design and enzyme design by automatic pinpointing potential active sites in available protein scaffolds. Proteins 80, 1078–1094 (2012).

  19. 19.

    , , , & Biochimie 88, 1631–1638 (2006).

  20. 20.

    & Methanobacterium thermoautotrophicus sp. n., an anaerobic, autotrophic, extreme thermophile. J. Bacteriol. 109, 707–715 (1972).

  21. 21.

    & Chemical forms of uranium in artificial seawater. J. Nucl. Sci. Technol. 19, 145–150 (1982).

  22. 22.

    & Characterization of a helical protein designed from first principles. Science 241, 976–978 (1988).

  23. 23.

    et al. Crystal structure of a synthetic triple-stranded alpha-helical bundle. Science 259, 1288–1293 (1993).

  24. 24.

    , , , & Kinetics of folding of leucine zipper domains. Biochemistry 34, 4097–4107 (1995).

  25. 25.

    & Artificial metalloenzymes: combining the best features of homogenous and enzymatic catalysis. Synlett 20, 3225–3236 (2009).

  26. 26.

    , , , & Lombardi, A. De novo design and structural characterization of proteins and metalloproteins. Annu. Rev. Biochem. 68, 779–819 (1999).

  27. 27.

    & Moser, C. C. Engineering enzymes. Faraday Discuss. 148, 443–448 (2011).

  28. 28.

    et al. Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis. Nature Chem. Biol. 8, 294–300 (2012).

  29. 29.

    et al. Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold. Science 334, 373–376 (2011).

  30. 30.

    , , & Expanding the utility of proteins as platforms for coordination chemistry. Coord. Chem. Rev. 225, 790–803 (2011).

  31. 31.

    et al. Metal-directed, chemically tunable assembly of one-, two- and three-dimensional crystalline protein arrays. Nature Chem. 4, 375–382 (2012).

  32. 32.

    Computational protein design: engineering molecular diversity, nonnatural enzymes, nonbiological cofactor complexes, and membrane proteins. Curr. Opin. Chem. Biol. 15, 452–457 (2011).

  33. 33.

    et al. Iterative approach to computational enzyme design. Proc. Natl Acad. Sci. USA 109, 3790–3795 (2012).

  34. 34.

    et al. Nonnatural protein–protein interaction-pair design by key residues grafting. Proc. Natl Acad. Sci. USA 104, 5330–5335 (2007).

  35. 35.

    et al. Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science 332, 816–821 (2011).

  36. 36.

    , , & Design of functional metalloproteins. Nature 460, 855–862 (2009).

  37. 37.

    , , & Identifying important structural characteristics of arsenic resistance proteins by using designed three stranded coils. Proc. Natl Acad. Sci. USA 104, 11969–11974 (2007).

Download references

Acknowledgements

This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy, under contract number DE-FG02-07ER15865 to C.H., and at Argonne National Laboratory (M.J.) under contract number DE-AC02-06CH11357, the Dreyfus Foundation Postdoctoral Program in Environmental Chemistry to S.O., the Ministry of Science and Technology of China (2009CB918500) and the National Natural Science Foundation of China (21173013, 11021463) to L.L. Use of the Advanced Photon Source for protein crystallography data collection at beamlines LS/CA-CAT (21-ID-F) and NE-CAT (24-ID-C) was supported by the Office of Basic Energy Sciences of the US Department of Energy under contract number DE-AC02-06CH11357. We thank S. F. Reichard for editing the manuscript and C. Yang and L. Lan for experimental support.

Author information

Author notes

    • Lu Zhou
    • , Mike Bosscher
    •  & Changsheng Zhang

    These authors contributed equally to this work

Affiliations

  1. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA

    • Lu Zhou
    • , Mike Bosscher
    • , Salih Özçubukçu
    • , Liang Zhang
    • , Wen Zhang
    • , Charles J. Li
    • , Jianzhao Liu
    •  & Chuan He
  2. BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering and Center for Quantitative Biology, Peking University, Beijing 100871, China

    • Changsheng Zhang
    •  & Luhua Lai
  3. Center for Life Sciences, Peking University, Beijing 100871, China

    • Changsheng Zhang
    •  & Luhua Lai
  4. Chemical Sciences and Engineering Division, Argonne National Laboratory, Argonne, Illinois 60439, USA

    • Mark P. Jensen

Authors

  1. Search for Lu Zhou in:

  2. Search for Mike Bosscher in:

  3. Search for Changsheng Zhang in:

  4. Search for Salih Özçubukçu in:

  5. Search for Liang Zhang in:

  6. Search for Wen Zhang in:

  7. Search for Charles J. Li in:

  8. Search for Jianzhao Liu in:

  9. Search for Mark P. Jensen in:

  10. Search for Luhua Lai in:

  11. Search for Chuan He in:

Contributions

C.H. conceived the project. C.H. and L.L. designed the experiment. C.Z. and L.L. performed the initial screening. W.Z. and C.J.L. performed subcloning of virtual hits. S.O. and C.J.L. expressed, purified and tested all first-generation virtual hits. L.Z., S.O. and M.B. designed second-generation mutants. L.Z., M.B. and J.L. expressed and tested all protein derivatives and designed all later-generation mutants. L.Z., M.B., M.P.J. and C.H. analysed the data and M.B. and L.Z. co-wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Luhua Lai or Chuan He.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    Supplementary information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchem.1856

Further reading