Advertisement

Nitric Oxide pp 183-203 | Cite as

Identification of NO-Sensitive Cysteine Residues Using Cysteine Mutants of Recombinant Proteins

  • Azam Shekariesfahlan
  • Christian Lindermayr
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)

Abstract

Nitric oxide (NO) is a free radical gas regulating a wide range of biological processes in plants. Proteins are the main reaction target of NO inside the cells. The relevance of S-nitrosation as one of the NO-mediated protein posttranslational modifications has been studied in detail. S-nitrosylation causes alterations of the activity/function, sub-cellular localization or interaction partners of proteins. Up to present, a large number of S-nitrosation candidates have been detected in plants. Recombinant proteins are widely used to show or confirm the protein posttranslational modifications. Here, using recombinant proteins subjected to biotin switch assay, the S-nitrosation of some nuclear candidates of Arabidopsis is verified. Proteins usually contain several cysteine residues which each might involve in structure of protein active sites. So, an important question is: which cysteine residue is the target of S-nitrosation and does it belong to an active site? Here, using the approach of substitution of cysteines by serines on recombinant proteins, the NO-sensitive cysteine residue of an Arabidopsis nuclear protein is identified. The next step could be to investigate the effect of S-nitrosation on protein activity/function and further to test the role of target cysteines and S-nitrosation of them in protein activity/function.

Key words

Nitric oxide S-nitrosation Biotin switch assay Arabidopsis nuclear proteins Recombinant proteins Cysteine mutation 

References

  1. 1.
    Lindermayr C, Sell S, Durner J (2008) Generation and detection of S-nitrosothiols. Methods Mol Biol 476:217–229PubMedGoogle Scholar
  2. 2.
    Kovacs I, Lindermayr C (2013) Nitric oxide-based protein modification: formation and site-specificity of protein S-nitrosylation. Front Plant Sci 4:137.  https://doi.org/10.3389/fpls.2013.00137 PubMedPubMedCentralGoogle Scholar
  3. 3.
    Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH (2001) Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol 3(2):193–197CrossRefPubMedGoogle Scholar
  4. 4.
    Lindermayr C, Saalbach G, Durner J (2005) Proteomic identification of S-nitrosylated proteins in Arabidopsis. Plant Physiol 137(3):921–930.  https://doi.org/10.1104/pp.104.058719 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1):207–234CrossRefPubMedGoogle Scholar
  6. 6.
    Chaki M, Shekariesfahlan A, Ageeva A, Mengel A, von Toerne C, Durner J, Lindermayr C (2015) Identification of nuclear target proteins for S-nitrosylation in pathogen-treated Arabidopsis thaliana cell cultures. Plant Sci 238:115–126.  https://doi.org/10.1016/j.plantsci.2015.06.011 CrossRefPubMedGoogle Scholar
  7. 7.
    Zheng L, Baumann U, Reymond JL (2004) An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32(14):e115.  https://doi.org/10.1093/nar/gnh110 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Azam Shekariesfahlan
    • 1
  • Christian Lindermayr
    • 2
  1. 1.Iranian Research Institute of Plant ProtectionAgricultural Research, Education and Extension Organization (AREEO)TehranIran
  2. 2.Helmholtz Zentrum München–German Research GmbH, Center for Environment HealthInstitute of Biochemical Plant Pathology, Ingolstädter Landstraße 1Munich-NeuherbergGermany

Personalised recommendations