Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Design, structure and stability of a hyperthermophilic protein variant

Nature Structural Biology volume 5, pages 470–475 (1998)Cite this article

Abstract

Here we report the use of an objective computer algorithm in the design of a hyperstable variant of the Streptococcal protein G β1 domain (Gβ1). The designed seven-fold mutant, Gβ1-c3b4, has a melting temperature in excess of 100 °C and an enhancement in thermodynamic stability of 4.3 kcal mol−1 at 50 °C over the wild-type protein. Gβ1-c3b4 maintains the Gβ1 fold, as determined by nuclear magnetic resonance spectroscopy, and also retains a significant level of binding to human IgG in qualitative comparisons with wild type. The basis of the stability enhancement appears to have multiple components including optimized core packing, increased burial of hydrophobic surface area, more favorable helix dipole interactions, and improvement of secondary structure propensity. The design algorithm is able to model such complex contributions simultaneously using empirical physical/chemical potential functions and a combinatorial optimization algorithm based on the dead-end elimination theorem. Because the design methodology is based on general principles, there is the potential of applying the methodology to the stabilization of other unrelated protein folds.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Lee, B. & Vasmatzis, G. Stabilization of protein structures. Curr. Opin. Biotech. 8, 423–428 (1997).

    Article  CAS  PubMed  Google Scholar 

  2. Straume, M., Murphy, K.P. & Freire, E. In Biocatalysis at extreme temperatures: enzyme systems near and above 100 °C Vol. 498 (eds Adams, M.W.W. & Kelly, R.M.) 122–135 (American Chemical Society, Washington, D.C., 1992).

    Book  Google Scholar 

  3. Rees, D.C. & Adams, M.W.W. Hyperthermophiles: taking the heat and loving it. Structure 3, 251–254 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Eidsness, M.K., Richie, K.A., Burden, A.E., Kurtz, J.D.M. & Scott, R.A. Dissecting contributions to the thermostability of pyrococcus furiosus rubredoxin: β-sheet chimeras. Biochemistry 36, 10406–10413 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Jiang, X., Bishop, E.J. & Farid, R.S. A de novo designed protein with properties that characterize natural hyperthermophilic proteins. J. Amer. Chem. Soc. 119, 838–839 (1997).

    Article  CAS  Google Scholar 

  6. Dahiyat, B.I. & Mayo, S.L. Protein design automation. Protein Sci. 5, 895–903 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Dahiyat, B.I., Gordon, D.B. & Mayo, S.L. Automated design of the surface positions of protein helices. Protein Sci. 6, 1333–1337 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Dahiyat, B.I. & Mayo, S.L. Probing the role of packing specificity in protein design. Proc. Watl. Acad. Sci. USA 94, 10172–10177 (1997).

    Article  CAS  Google Scholar 

  9. Dahiyat, B.I., Sarisky, C.A. & Mayo, S.L. De novo protein design: towards fully automated sequence selection. J. Mol. Biol. 273, 789–796 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Dahiyat, B.I. & Mayo, S.L. De novo protein design: fully automated sequence selection. Science 278, 82–87 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. Desmet, J., De Maeyer, M., Hazes, B. & Lasters, I. The dead-end elimination theorem and its use in protein side-chain positioning. Nature 356, 539–542 (1992).

    Article  CAS  PubMed  Google Scholar 

  12. Goldstein, R.F. Efficient rotamer elimination applied to protein side-chains and related spin-glasses. Biophys. J. 66, 1335–1340 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. De Maeyer, M., Desmet, J. & Lasters, I. All in one: a highly detailed rotamer library improves both accuracy and speed in the modeling of sidechains by dead-end elimination. Folding & Design 2, 53–66 (1997).

    Article  CAS  Google Scholar 

  14. Gronenborn, A.M. et al. A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science 253, 657–661 (1991).

    Article  CAS  PubMed  Google Scholar 

  15. Gallagher, T., Alexander, P., Bryan, P. & Gilliland, G.L. Two crystal structures of the β1 immunoglobulin-binding domain of streptococcal protein G and comparison with NMR. Biochemistry 33, 4721–4729 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Bernstein, F.C. et al. The Protein Data Bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535–542 (1977).

    Article  CAS  PubMed  Google Scholar 

  17. Su, A. & Mayo, S.L. Coupling backbone flexibility and amino acid sequence selection in protein design. Protein Sci. 6, 1701–1707 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Janin, J., Wodak, S., Levitt, M. & Maigret, B. Conformation of amino acid side-chains in proteins. J. Mol. Biol. 125, 357–386 (1978).

    Article  CAS  PubMed  Google Scholar 

  19. Ponder, J.W. & Richards, F.M. Tertiary templates for proteins-use of packing criteria in the enumeration of allowed sequences for different structural classes. J. Mol. Biol. 193, 775–791 (1987).

    Article  CAS  PubMed  Google Scholar 

  20. Dunbrack, R.L. & Karplus, M. Backbone dependent rotamer library for proteins - an application to side-chain prediction. J. Mol. Biol. 230, 543–574 (1993).

    Article  CAS  PubMed  Google Scholar 

  21. Zhang, X.J., Baase, W.A., Shoichet, B.K., Wilson, K.P. & Matthews, B.W. Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive. Prot. Engng. 8, 1017–1022 (1995).

    Article  CAS  Google Scholar 

  22. Serrano, L., Day, A.G. & Fersht, A.R. Step-wise mutation of barnase to binase: A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. J. Mol, Biol. 233, 305–312 (1993).

    Article  CAS  Google Scholar 

  23. Pantoliano, M.W. et al. Large increases in general stability for subtilisin BPN′ through incremental changes in the free energy of unfolding. Biochemistry 28, 7205–7213 (1989).

    Article  CAS  PubMed  Google Scholar 

  24. Jeener, J., Meier, B.H., Bachmann, P. & Ernst, R.R. Investigation of exchange processes by two-dimensional NMR spectroscopy. J. Chem. Phys. 71, 4546–4553 (1979).

    Article  CAS  Google Scholar 

  25. Piantini, U., Sorensen, O.W. & Ernst, R.R. Multiple quantum filters for elucidating NMR coupling networks. J. Amer. Chem. Soc. 104, 6800–6801 (1982).

    Article  CAS  Google Scholar 

  26. Bax, A. & Davis, D.G. MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65, 355–360 (1985).

    CAS  Google Scholar 

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

    Book  Google Scholar 

  28. Brünger, A.T. X-PLOR, version 3.1, a system for X-ray crystallography and NMR, (Yale Univ. Press, New Haven, CT, 1992).

    Google Scholar 

  29. Nilges, M., Clore, G.M. & Gronenborn, A.M. Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry- dynamical simulated annealing calculations. FEBS Lett. 229, 317–324 (1988).

    Article  CAS  PubMed  Google Scholar 

  30. Nilges, M., Kuszewski, J. & Brünger, A.T. Sampling properties of simulated annealing and distance geometry. In Computational Aspects of the Study of Biological Macromolecules by NMR (eds Hoch, J.C., Poulsen, F.M. & Redfield, C.) 451–457 (Plenum Press, New York, 1991).

    Google Scholar 

  31. Kuszewski, J., Nilges, M. & Brünger, A.T. Sampling and efficiency of metric matrix distance geometry — a novel partial metrization algorithm. J. Biomol. NMR 2, 33–56 (1992).

    Article  CAS  PubMed  Google Scholar 

  32. Hyberts, S.G., Goldberg, M.S., Havel, T.F. & Wagner, G. The solution structure of eglin-c based on measurements of many NOEs and coupling-constants and its comparison with X-ray structures. Protein Sci. 1, 736–751 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  34. Gronenborn, A.M. & Clore, G.M. Identification of the contact surface of a streptococcal protein G-domain complexed with a human Fc fragment. J. Mol. Biol. 233, 331–335 (1993).

    Article  CAS  PubMed  Google Scholar 

  35. Huyghues-Despointes, B.M.P., Scholtz, J.M. & Baldwin, R.L. Effect of a single aspartate on helix stability at different positions in a neutral alanine based peptide. Protein Sci. 2, 1604–1611 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Rohl, C.A., Chakrabartty, A. & Baldwin, R.L. Helix propagation and N-cap propensities of the amino acids measured in alanine-based peptides in 40 volume percent trifluoroethanol. Protein Sci. 5, 2623–2637 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sun, S., Brem, R., Chan, H.S. & Dill, K.A. Designing amino acid sequences to fold with good hydrophobic cores. Protein Engng. 8, 1205–1213 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  40. 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  PubMed  Google Scholar 

  41. Alexander, P., Fahnestock, S., Lee, T., Orban, J. & Bryan, P. Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains β1 and β2 — why small proteins tend to have high denaturation temperatures. Biochemistry 31, 3597–3603 (1992).

    Article  CAS  PubMed  Google Scholar 

  42. Johnson, B.H. & Hecht, M.H. Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Biotechnology 12, 1357–1360 (1994).

    CAS  PubMed  Google Scholar 

  43. Santoro, M.M. & Bolen, D.W. Unfolding free-energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27, 8063–8068 (1988).

    Article  CAS  PubMed  Google Scholar 

  44. Piotto, M., Saudek, V. & Sklenar, V. Gradient tailored excitation for single quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2, 661–665 (1992).

    Article  CAS  PubMed  Google Scholar 

  45. Kraulis, P.J. ANSIG: a program for the assignment of protein 1H 2D NMR spectra by interactive computer graphics. J. Magn. Reson. 84, 627–633 (1989).

    CAS  Google Scholar 

  46. Driscoll, P.C., Gronenborn, A.M., Wingfield, P.T. & Clore, G.M. Determination of the secondary structure and molecular topology of interleukin-1-β by use of 2-dimensional and 3-dimensional heteronuclear 1H-15N NMR spectroscopy. Biochemistry 29, 4668–4682 (1990).

    Article  CAS  PubMed  Google Scholar 

  47. Koradi, R., Billeter, M. & Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–Continues (1996).

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Biology, California Institute of Technology, mail code 147-75, Pasadena, California, 91125, USA

    Sandra M. Malakauskas

  2. Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, mail code 147-75, Pasadena, California, 91125, USA

    Stephen L. Mayo

Authors
  1. Sandra M. Malakauskas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen L. Mayo
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Malakauskas, S., Mayo, S. Design, structure and stability of a hyperthermophilic protein variant. Nat Struct Mol Biol 5, 470–475 (1998). https://doi.org/10.1038/nsb0698-470

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb0698-470

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing