Skip to main content
SpringerLink
Log in

Protein Disulfide Bonds Detected by Tagging with High Molecular Weight Maleimide Derivative

  • STRUCTURAL–FUNCTIONAL ANALYSIS OF BIOPOLYMERS AND THEIR COMPLEXES
  • Published:
Molecular Biology Aims and scope Submit manuscript

Abstract

Disulfide bridges are essential for maintaining the structure and function of proteins. Traditionally, studies of the disulfide bonds require expensive equipment and high purity of the protein sample, therefore, the development of simpler techniques is warranted. Here, were present a novel protocol for the detection of disulfide bonds in proteins, which is based on the labeling reduced disulfide bridges with a high molecular weight (HMW) maleimide derivative. After irreversible blocking of free thiol groups of proteins, the labeling of new thiols released from disulfide bridges with a high-molecular-weight (HMW) maleimide derivative is performed. To confirm localization of cysteines involved in the formation of disulfide bonds, cysteine mutagenesis was conducted. For validation, aquaporin 5 (AQP5) and transient receptor potential cation channel subfamily V member 4 (TRPV4) proteins were tagged with FLAG (DYKDDDDK) on N-termini. Increase in MW of the target proteins from immunoblot indicated the presence of disulfide bonds. No bands with increased MW were detected in AQP5, while TPRV4 cysteines at disulfide bridges-constituting positions 639, 645, 652, 660, 770 were detected and confirmed by cysteine mutagenesis. These data indicate that the proposed technique is feasible and effective for the detection of protein disulfide bonds.

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

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.

Similar content being viewed by others

REFERENCES

  1. Bulaj G. 2005. Formation of disulfide bonds in proteins and peptides. Biotechnol. Adv. 23, 87–92.

    Article  CAS  Google Scholar 

  2. Cook K.M., Hogg P.J. 2013. Post-translational control of protein function by disulfide bond cleavage. Antioxid. Redox Signal. 18, 1987–2015.

    Article  CAS  Google Scholar 

  3. Goldberger R.F., Epstein C.J., Anfinsen C.B. 1963. Acceleration of reactivation of reduced bovine pancreatic ribonuclease by a microsomal system from rat liver. J. Biol. Chem. 238, 628–635.

    Article  CAS  Google Scholar 

  4. Anfinsen C.B., Haber E. 1961. Studies on the reduction and re-formation of protein disulfide bonds. J. Biol. Chem. 236, 1361–1363.

    Article  CAS  Google Scholar 

  5. Bonander N., Leckner J., Guo H., Karlsson B.G., Sjolin L. 2000. Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: How important is the S–S bond for folding and stability? Eur. J. Biochem. 267, 4511–4519.

    CAS  PubMed  Google Scholar 

  6. Wetzel R., Perry L.J., Baase W.A., Becktel W.J. 1988. Disulfide bonds and thermal stability in T4 lysozyme. Proc. Natl. Acad. Sci. U. S. A. 85, 401–405.

    Article  CAS  Google Scholar 

  7. Hogg P.J. 2003. Disulfide bonds as switches for protein function. Trends Biochem. Sci. 28, 210–214.

    Article  CAS  Google Scholar 

  8. Go Y.M., Jones D.P. 2013. Thiol/disulfide redox states in signaling and sensing. Crit. Rev. Biochem. Mol. Biol. 48, 173–181.

    Article  CAS  Google Scholar 

  9. Tarnow P., Schoneberg T., Krude H., Gruters A., Biebermann H. 2003. Mutationally induced disulfide bond formation within the third extracellular loop causes melanocortin 4 receptor inactivation in patients with obesity. J. Biol.Chem. 278, 48666-48673.

    Article  CAS  Google Scholar 

  10. Singh R. 2008. A review of algorithmic techniques for disulfide-bond determination. Brief. Funct. Genomics Proteomics. 7, 157–172.

    Article  CAS  Google Scholar 

  11. Tsai C.H., Chan C.H., Chen B.J., Kao C.Y., Liu H.L., Hsu J.P. 2007. Bioinformatics approaches for disulfide connectivity prediction. Curr. Prot. Peptide Sci. 8, 243–260.

    Article  CAS  Google Scholar 

  12. Singh R., Murad W. 2013. Protein disulfide topology determination through the fusion of mass spectrometric analysis and sequence-based prediction using Dempster–Shafer theory. BMC Bioinformatics. 14 (Suppl. 2), S20.

    Article  CAS  Google Scholar 

  13. Planey S.L. 2012. Discovery of selective and potent inhibitors of palmitoylation. InTech. 9, 251–288.

    Google Scholar 

  14. Fontaine S.D., Reid R., Robinson L., Ashley G.W., Santi D.V. 2015. Long-term stabilization of maleimide-thiol conjugates. Bioconj. Chem. 26, 145–152.

    Article  CAS  Google Scholar 

  15. Ravi S., Krishnamurthy V.R., Caves J.M., Haller C.A., Chaikof E.L. 2012. Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer. Acta Biomater. 8, 627–635.

    Article  CAS  Google Scholar 

  16. Ghosh S.S., Kao P.M., McCue A.W., Chappelle H.L. 1990. Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes. Bioconj. Chem. 1, 71–76.

    Article  CAS  Google Scholar 

  17. Singh R., Whitesides G.M. 1994. Reagents for rapid reduction of native disulfide bonds in proteins. Bioorg. Chem. 22, 109–115.

    Article  CAS  Google Scholar 

  18. Ding Q.W., Zhang Y., Wang Y., Wang Y.N., Zhang L., Ding C., Wu L.L., Yu G.Y. 2010. Functional vanilloid receptor-1 in human submandibular glands. J. Dental Res. 89, 711–716.

    Article  CAS  Google Scholar 

  19. Nordlund H.R., Laitinen O.H., Uotila S.T., Nyholm T., Hytonen V.P., Slotte J.P., Kulomaa M.S. 2003. Enhancing the thermal stability of avidin. Introduction of disulfide bridges between subunit interfaces. J. Biol. Chem. 278, 2479–2483.

    Article  CAS  Google Scholar 

  20. Carugo O., Cemazar M., Zahariev S., Hudaky I., Gaspari Z., Perczel A., Pongor S. 2003. Vicinal disulfide turns. Prot. Eng. 16, 637–639.

    Article  CAS  Google Scholar 

  21. Caterina M.J. 2007. Transient receptor potential ion channels as participants in thermosensation and thermoregulation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R64–R76.

    Article  CAS  Google Scholar 

  22. Wang S., Chuang H.H. 2011. C-terminal dimerization activates the nociceptive transduction channel transient receptor potential vanilloid 1. J. Biol. Chem. 286, 40601–4067.

    Article  CAS  Google Scholar 

  23. Deng Z., Paknejad N., Maksaev G., Sala-Rabanal M., Nichols C.G., Hite R.K., Yuan P. 2018. Cryo-EM and X-ray structures of TRPV4 reveal insight into ion permeation and gating mechanisms. Nat. Struct. Mol. Biol. 25, 252–260.

    Article  CAS  Google Scholar 

  24. Duan J., Li J., Zeng B., Chen G.L., Peng X., Zhang Y., Wang J., Clapham D.E., Li Z., Zhang J. 2018. Structure of the mouse TRPC4 ion channel. Nat. Commun. 9, 3102.

    Article  Google Scholar 

Download references

ACKNOWLEDGMENTS

We are grateful to Prof. Miguel A. Valverde (Universitat Pompeu Fabra, Barcelona, Spain) for his generous gifts of plasmids and to Prof. Xiaomin Wang (Capital Medical University, Beijing, China) for assistance with the lab equipment.

Funding

This study was supported by grants from the National Nature Science Foundation of China (nos. 81100765, 81570990).

Author information

Authors and Affiliations

  1. Beijing Stomatological Hospital and School of Stomatology, Capital Medical University, 100050, Beijing, China

    Q. W. Ding

  2. Department of Stomatology, Beijing Chao-Yang Hospital, Capital Medical University, 100020, Beijing, China

    M. Lin

Authors
  1. Q. W. Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The text was submitted by the author(s) in English.

Corresponding author

Correspondence to Q. W. Ding.

Ethics declarations

The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ding, Q.W., Lin, M. Protein Disulfide Bonds Detected by Tagging with High Molecular Weight Maleimide Derivative. Mol Biol 55, 449–457 (2021). https://doi.org/10.1134/S0026893321020187

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0026893321020187

Keywords:

Search

Navigation