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Novel metal-binding proteins by design

Nature Structural Biology volume 2, pages 368–373 (1995)Cite this article

A Corrigendum to this article was published on 01 October 1995

Abstract

We describe the successful design of a tetrahedral His3Cys Zn(II)-binding site in a small protein of known structure: the B1 domain of Streptococcal protein G. The B1 variants containing the novel metal-binding site were characterized using a combination of optical absorption, circular dichroism and NMR spectroscopies. The results indicate that the designed proteins bind Zn(II) with high affinity and tetrahedral coordination geometry, and that the overall secondary and tertiary structure of the B1 domain is maintained.

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References

  1. Regan, L. The design of metal-binding sites in proteins. A. Rev. Biophys. biomol. Struct. 22, 257–281 (1993).

    Article  CAS  Google Scholar 

  2. Corey, D.R. & Schultz, P.G. Introduction of a metal-dependent regulatory switch into an enzyme. J. biol. Chem. 264, 3666–3669 (1989).

    CAS  PubMed  Google Scholar 

  3. Higaki, J.N., Haymore, B.L., Chen, S., Fletterick, R.J. & Craik, C.S. Regulation of serine protease activity by an engineered metal switch. Biochemistry 29, 8582–8586 (1990).

    Article  CAS  Google Scholar 

  4. Kellis, J.T., Jr, Todd, R. J. & Arnold, F.H. Protein stabilization by engineered metal chelation. BioTechnology 9, 994–995 (1991).

    Article  CAS  Google Scholar 

  5. Ghadiri, M.R., Soares, C. & Choi, C. Design of an artificial four-helix bundle metalloprotein via a novel ruthenium(II)-assisted self-assembly process. J. Am. chem. Soc. 114, 4000–1002 (1992).

    Article  CAS  Google Scholar 

  6. Iverson, B.L. et al. Metalloantibodies. Science 249, 659–662 (1990).

    Article  CAS  Google Scholar 

  7. 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 

  8. Pessi, A. et al. A designed metal-binding protein with a novel fold. Nature 362, 367–369 (1993).

    Article  CAS  Google Scholar 

  9. Regan, L. & Clarke, N.D. Atetrahedral zinc(II)-binding site introduced into a designed protein. Biochemistry 29, 10878–10883 (1990).

    Article  CAS  Google Scholar 

  10. Gronenborn, A.M. et al. The immunoglobin binding domain of Streptococcal Protein G has a novel and highly stable polypeptide fold. Science 253, 657–661 (1991).

    Article  CAS  Google Scholar 

  11. Brünger, A.T. X-PLOR Version 3.1: A system for X-ray crystallography and NMR (Yale University Press, New Haven, 1992).

    Google Scholar 

  12. Smith, C.K., Withka, J.M. & Regan, L. A thermodynamic scale for the β-sheet forming tendencies of the amino acids. Biochemistry 33, 5510–5517 (1994).

    Article  CAS  Google Scholar 

  13. Bertini, I. & Luchinat, C. High spin cobalt(II) as a probe for the investigation of metalloproteins. Adv. inorg. Chem. 6, 71–111 (1984).

    CAS  Google Scholar 

  14. Krizek, B.A., Merkle, D.L. & Berg, J.M. Ligand variation and metal ion binding specificity in zinc finger proteins. Inorg. Chem. 32, 937–940 (1993).

    Article  CAS  Google Scholar 

  15. Lippard, S.J. & Berg, J.M. Principles of Bioinorganic Chemistry 214–215 (University Science Books, Mill Valley, CA, 1994).

    Google Scholar 

  16. Berg, J.M. & Merkle, D.L. On the metal ion specificity of “zinc finger” proteins. J Am. chem. Soc. 111, 3759–3761 (1989).

    Article  CAS  Google Scholar 

  17. Alexander, P., Orban, J. & Bryan, P. Kinetic analysis of folding and unfolding the 56 amino acid IgG-binding domain of Streptococcal Protein G. Biochemistry 31, 7243–7248 (1992).

    Article  CAS  Google Scholar 

  18. Brahms, S. & Brahms, J. Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J. molec. Biol. 138, 149–178 (1980).

    Article  CAS  Google Scholar 

  19. Frankel, A.D., Berg, J.M. & Pabo, C.O. Metal-dependent folding of a single zinc finger from transcription factor IIIA. Proc. natn. Acad. Sci. U.S.A. 84, 4841–4845 (1987).

    Article  CAS  Google Scholar 

  20. Párraga, G. et al, Zinc-dependent structure of a single-finger domain of yeast ADR1. Science 241, 1489–1492 (1988).

    Article  Google Scholar 

  21. Coleman, J.E. Cadmium-113 nuclear magnetic resonance applied to metalloproteins. Meth. Enzymol. 227, 16–43 (1993).

    Article  CAS  Google Scholar 

  22. Kim, C.A. & Berg, J.M. Thermodynamic β-sheet propensities measured using a zinc-finger host peptide. Nature 362, 267–270 (1993).

    Article  CAS  Google Scholar 

  23. Minor, D.L. Jr & Kim, P.S. Measurement of the β-sheet-forming propensities of the amino acids. Nature 367, 660–663 (1994).

    Article  CAS  Google Scholar 

  24. Vallee, B.L. & Auld, D.S. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647–5659 (1990).

    Article  CAS  Google Scholar 

  25. Lee, B.-K. & Richards, F.M. The interpretation of protein structures: estimation of static accessibility. J. molec. Biol. 55, 397–400 (1971).

    Article  Google Scholar 

  26. Maniatis, T., Fritsch, E.F. & Sambrook, J. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982).

    Google Scholar 

  27. Riddles, P.W., Blakeley, R.L. & Zerner, B. Reassessment of Ellman's reagent. Meth. Enzymol. 91, 49–60 (1983).

    Article  CAS  Google Scholar 

  28. Wüthrich, K. NMR of Proteins and Nucleic Acids (John Wiley, New York, 1986).

    Book  Google Scholar 

  29. Carson, M. Ribbons 2.2 Manual, (University of Alabama at Birmingham, Birmingham, 1993).

    Google Scholar 

  30. Kraulis, P. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  31. Bagshaw, C.R. & Harris, D.A. in Spectrophotometry and Spectrofluorimetry: A Practical Approach (eds Bashford, C. L. & Harris, D. A.) 91–113 (IRL Press, Washington, 1987).

    Google Scholar 

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Authors and Affiliations

  1. Department of Molecular Biophysics & Biochemistry Yale University, 266 Whitney Avenue, New Haven, Connecticut, 06520, USA

    Michael Klemba, Kevin H. Gardner, Stephen Marino & Lynne Regan

  2. Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, 21205, USA

    Neil D. Clarke

Authors
  1. Michael Klemba
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  2. Kevin H. Gardner
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  3. Stephen Marino
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  4. Neil D. Clarke
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  5. Lynne Regan
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Klemba, M., Gardner, K., Marino, S. et al. Novel metal-binding proteins by design. Nat Struct Mol Biol 2, 368–373 (1995). https://doi.org/10.1038/nsb0595-368

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