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.

  • Letter
  • Published:

Design of single-layer β-sheets without a hydrophobic core

Nature volume 403, pages 456–460 (2000)Cite this article

Abstract

The hydrophobic effect is the main thermodynamic driving force in the folding of water-soluble proteins1,2. Exclusion of nonpolar moieties from aqueous solvent results in the formation of a hydrophobic core in a protein, which has been generally considered essential for specifying and stabilizing the folded structures of proteins1,2,3,4,5,6. Outer surface protein A (OspA) from Borrelia burgdorferi contains a three-stranded β-sheet segment which connects two globular domains7. Although this single-layer β-sheet segment is exposed to solvent on both faces and thus does not contain a hydrophobic core, the segment has a high conformational stability8. Here we report the engineering of OspA variants that contain larger single-layer β-sheets (comprising five and seven β-strands) by duplicating a β-hairpin unit within the β-sheet. Nuclear magnetic resonance and small-angle X-ray scattering analyses reveal that these extended single-layer β-sheets are formed as designed, and amide hydrogen–deuterium exchange and chemical denaturation show that they are stable. Thus, interactions within the β-hairpin unit and those between adjacent units, which do not involve the formation of a hydrophobic core, are sufficient to specify and stabilize the single-layer β-sheet structure. Our results provide an expanded view of protein folding, misfolding and design.

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

Figure 1: Structure of wild-type OspA.
Figure 2: The β-sheet extension design in OspA+1bh.
Figure 3: NMR characterization of the extended single-layer β-sheet in OspA+1bh.
Figure 4: Nitrogen-15 T1/T2 ratios of the amide 15N nuclei in OspA+1bh plotted against the angle θ between the N-H bond vectors and the estimated unique axis of the rotational diffusion tensor.
Figure 5: The P(r) functions of OspA+1bh (a) and OspA+2bh (b) determined from SAXS data.
Figure 6: Urea-induced unfolding of wild-type OspA (open circles), OspA+1bh (open triangles), OspA+2bh (crosses), OspA mutant F126A (filled circles) and OspA+1bh mutant K119′T/F126′L/K135′G (closed triangles).

Similar content being viewed by others

References

  1. Kauzmann, W. Some factors in the interpretation of protein denaturation. Adv. Prot. Chem. 14, 1–63 ( 1959).

    CAS  Google Scholar 

  2. Dill, K. A. Dominant forces in protein folding. Biochemistry 29 , 7133–7155 (1990).

    Article  CAS  Google Scholar 

  3. Harbury, P. B., Plecs, J. J., Tidor, B., Alber, T. & Kim, P. S. High-resolution protein design with backbone freedom. Science 282, 1462–1467 (1998).

    Article  CAS  Google Scholar 

  4. Beasley, J. R. & Hecht, M. H. Protein design: the choice of de novo sequences. J. Biol. Chem. 272, 2031 –2034 (1997).

    Article  CAS  Google Scholar 

  5. Bryson, J. W. et al. Protein design: a hierarchic approach. Science 270, 935–941 ( 1995).

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Li, H., Dunn, J. J., Luft, B. J. & Lawson, C. L. Crystal structure of Lyme disease antigen outer surface protein A complexed with an Fab. Proc. Natl Acad. Sci. USA 94, 3584– 3589 (1997).

    Article  ADS  CAS  Google Scholar 

  8. Pham, T.-N., Koide, A. & Koide, S. A stable single-layer β-sheet without a hydrophobic core. Nature Struct. Biol. 5, 115–119 (1998).

    Article  CAS  Google Scholar 

  9. Bu, Z., Koide, S. & Engelman, D. M. A solution SAXS study of Borrelia burgdorferi OspA, a protein containing a single-layer β-sheet. Protein Sci. 7, 2681–2683 ( 1998).

    Article  CAS  Google Scholar 

  10. Pham, T. N. & Koide, S. NMR studies of Borrelia burgdorferi OspA, a 28 kDa protein containing a single-layer β-sheet. J. Biomol. NMR 11, 407– 414 (1998).

    Article  CAS  Google Scholar 

  11. Koide, S. et al. Multi-step denaturation of Borrelia burgdorferi OspA, a protein containing a single-layer β-sheet. Biochemistry 38, 4757–4767 ( 1999).

    Article  CAS  Google Scholar 

  12. Wishart, D. S. & Sykes, B. D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 4, 171–180 (1994).

    Article  CAS  Google Scholar 

  13. Kay, L. E., Torchia, D. A. & Bax, A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972–8979 (1989).

    Article  CAS  Google Scholar 

  14. Tjandra, N., Garrett, D. S., Gronenborn, A. M., Bax, A. & Clore, G. M. Defining long range order in NMR structure determination from the dependence of heteronuclear relaxation times on rotational diffusion anisotropy. Nature Struct. Biol. 4, 443–449 (1997).

    Article  CAS  Google Scholar 

  15. Lee, L. K., Rance, M., Chazin, W. J. & Palmer, A. G. Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13Cα nuclear spin relaxation. J. Biomol. NMR 9, 287–298 (1997).

    Article  CAS  Google Scholar 

  16. Kataoka, M., Kuwajima, K., Tokunaga, F. & Goto, Y. Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering. Protein Sci. 6, 422–430 (1997).

    Article  CAS  Google Scholar 

  17. Lattman, E. E. Small angle scattering studies of protein folding. Curr. Opin. Struct. Biol. 4, 87–92 ( 1994).

    Article  CAS  Google Scholar 

  18. Englander, S. W. & Kallenbach, N. R. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q. Rev. Biophys. 6, 521–655 (1984).

    Google Scholar 

  19. Hughson, F. M., Wright, P. E. & Baldwin, R. L. Structural characterization of a partly folded apomyoglobin intermediate. Science 249, 1544– 1548 (1990).

    Article  ADS  CAS  Google Scholar 

  20. Myers, J. K., Pace, C. N. & Scholtz, J. M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4, 2138–2148 ( 1995).

    Article  CAS  Google Scholar 

  21. Lacroix, E., Kortemme, T., de la Paz, M. L. & Serrano, L. The design of linear peptides that fold as monomeric beta-sheet structures. Curr. Opin. Struct. Biol. 9, 487– 493 (1999).

    Article  CAS  Google Scholar 

  22. Macias, M. J. et al. Structure of the WW domain of a kinase-associated protein complexed with a proline-rich peptide. Nature 382, 646–649 (1996).

    Article  ADS  CAS  Google Scholar 

  23. Koepf, E. K., Petrassi, H. M., Sudol, M. & Kelly, J. W. WW: An isolated three-stranded antiparallel beta-sheet domain that unfolds and refolds reversibly; evidence for a structured hydrophobic cluster in urea and GdnHCl and a disordered thermal unfolded state. Protein Sci. 8, 841–853 (1999).

    Article  CAS  Google Scholar 

  24. Fink, A. L. Protein aggregation: folding aggregates, inclusion bodies and amyloid. Fold. Des. 3, R9–R23 ( 1998).

    Article  CAS  Google Scholar 

  25. Kelly, J. W. The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr. Opin. Struct. Biol. 8, 101–106 (1998).

    Article  CAS  Google Scholar 

  26. Perutz, M. F., Johnson, T., Suzuki, M. & Finch, J. T. Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc. Natl Acad. Sci. USA 91, 5355– 5358 (1994).

    Article  ADS  CAS  Google Scholar 

  27. Clore, G. M. & Gronenborn, A. M. Applications of three- and four-dimensional heteronuclear NMR spectroscopy to protein structure determination. Prog. NMR Spectrosc. 23, 43– 92 (1991).

    Article  CAS  Google Scholar 

  28. Farrow, N. A. et al. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003 (1994).

    Article  CAS  Google Scholar 

  29. Brunger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

Download references

Acknowledgements

We thank B. M. Goldstein and J. J. Hayes for discussions; F. Delaglio, B. Johnson, L. E. Kay and A. G. Palmer III for NMR programs; and S. D. Kennedy for technical support. This work was supported in part by NIH grants to S.K. and D.M.E.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics University of Rochester Medical Center, Rochester, 14642, New York, USA

    Shohei Koide, Xiaolin Huang, Karl Link & Akiko Koide

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 06511, Connecticut , USA

    Zimei Bu & Donald M. Engelman

Authors
  1. Shohei Koide
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaolin Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Akiko Koide
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zimei Bu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Donald M. Engelman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shohei Koide.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Koide, S., Huang, X., Link, K. et al. Design of single-layer β-sheets without a hydrophobic core. Nature 403, 456–460 (2000). https://doi.org/10.1038/35000255

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35000255

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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