Skip to main content
SpringerLink
Log in

Detecting native folds in mixtures of proteins that contain disulfide bonds

  • Brief Communication
  • Published:

From Nature Biotechnology

View current issue Submit your manuscript

Abstract

High-throughput in vitro refolding of proteins that contain disulfide bonds, for which soluble expression is particularly difficult, is severely impeded by the absence of effective methods for detecting their native forms. We demonstrate such a method, which combines mass spectrometry with mild reductions, requires no prior experimentation or knowledge of proteins' physicochemical characteristics, function or activity, and is amenable to automation. These are necessary criteria for structural genomics and proteomics applications.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: ESI/FTMS mass spectra of seven multiple-disulfide-bond–containing proteins (a) The regeneration mixture, 14 h after initiation of oxidative folding (50 mM DTTox, 10 mM Ca2+, 20 mM Tris-HCl, pH 8) that was subjected to a reduction pulse, followed by AEMTS blocking.

Similar content being viewed by others

References

  1. Bhattacharya, A., Tejero, R. & Montelione, G.T. Proteins 66, 778–795 (2007).

    Article  CAS  Google Scholar 

  2. Fiser, A. & Simon, I. Methods Enzymol. 353, 10–21 (2002).

    Article  CAS  Google Scholar 

  3. Song, J.N., Wang, M.L., Li, W.J. & Xu, W.B . Biochem. Biophys. Res. Commun. 318, 142–147 (2004).

    Article  CAS  Google Scholar 

  4. Wedemeyer, W.J., Welker, E., Narayan, M. & Scheraga, H.A . Biochemistry 39, 4207–4216 (2000).

    Article  CAS  Google Scholar 

  5. Narayan, M., Welker, E., Wedemeyer, W.J. & Scheraga, H.A Acc. Chem. Res. 33, 805–812 (2000).

    Article  CAS  Google Scholar 

  6. Tu, B.P. & Weissman, J.S. J. Cell Biol. 164, 341–346 (2004).

    Article  CAS  Google Scholar 

  7. Wedemeyer, W.J., Xu, X., Welker, E. & Scheraga, H.A. Biochemistry 41, 1483–1491 (2002).

    Article  CAS  Google Scholar 

  8. Rothwarf, D.M., Li, Y.J. & Scheraga, H.A. Biochemistry 37, 3760–3766 (1998).

    Article  CAS  Google Scholar 

  9. Ruoppolo, M. et al. Biochemistry 39, 12033–12042 (2000).

    Article  CAS  Google Scholar 

  10. Welker, E., Hathaway, L. & Scheraga, H.A. J. Am. Chem. Soc. 126, 3720–3721 (2004).

    Article  CAS  Google Scholar 

  11. Arolas, J.L., Aviles, F.X., Chang, J.Y. & Ventura, S. Trends Biochem. Sci. 31, 292–301 (2006).

    Article  CAS  Google Scholar 

  12. van den Berg, B. et al. EMBO J. 18, 4794–4803 (1999).

    Article  CAS  Google Scholar 

  13. Ge, Y. et al. J. Am. Chem. Soc. 124, 672–678 (2002).

    Article  CAS  Google Scholar 

  14. Han, X., Jin, M., Breuker, K. & McLafferty, F.W. Science 314, 109–112 (2006).

    Article  CAS  Google Scholar 

  15. Welker, E., Narayan, M., Wedemeyer, W.J. & Scheraga, H.A. Proc. Natl. Acad. Sci. USA 98, 2312–2316 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by National Institutes of Health grants GM-24893 (to H.A.S.) and GM-16609 (to F.W.M.), by NORT(DNT), Hungary, OTKA-NF61431 (to E.W.), and by UTEP startup money (to M.N.). E.W. is a Howard Hughes Medical Institute international scholar and an EMBO-HHMI start up grantee.

Author information

Author notes

  1. Mahesh Narayan and Ervin Welker: These authors contributed equally to this work.

Authors and Affiliations

  1. Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, 14353-1301, New York, USA

    Mahesh Narayan, Ervin Welker, Huili Zhai, Xuemei Han, Guoqiang Xu, Fred W McLafferty & Harold A Scheraga

  2. Department of Chemistry, University of Texas at El Paso, El Paso, Texas, 79968, USA

    Mahesh Narayan

  3. Institute of Biochemistry, Biological Research Centre of the Hungarian Academy, H-6701, Szeged, 62 Temesvari krt., Hungary

    Ervin Welker

  4. Institute of Enzymology, Biological Research Centre of the Hungarian Academy, H-1114, Budapest, 29 Karolina ut., Hungary

    Ervin Welker

Authors
  1. Mahesh Narayan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ervin Welker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huili Zhai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xuemei Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guoqiang Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fred W McLafferty
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Harold A Scheraga
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Mahesh Narayan, Ervin Welker or Harold A Scheraga.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1,2, Notes, Methods (PDF 189 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Narayan, M., Welker, E., Zhai, H. et al. Detecting native folds in mixtures of proteins that contain disulfide bonds. Nat Biotechnol 26, 427–429 (2008). https://doi.org/10.1038/nbt1380

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1380

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation