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Synthesis and utilization of reversible and irreversible light-activated nanovalves derived from the channel protein MscL

Nature Protocols volume 2, pages 14261437 (2007) | Download Citation

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Abstract

This protocol details the chemical modification of the mechanosensitive channel of large-conductance (MscL) channel protein into a light-activated nanovalve and its utilization for triggered delivery in synthetic liposomal vesicles. It is based on charge-induced activation of this otherwise mechanosensitive channel by covalent attachment to the protein of rationally designed synthetic functionalities. In the dark, these functionalities will be uncharged and the channel will stay closed, but UV illumination will cause their ionization and trigger channel activity. In the case of reversible activation, subsequent illumination with visible light will neutralize the charge, causing the channel to close. The protocol includes synthesis of light-responsive compounds, protein isolation and its chemical labeling, reconstitution of the protein into artificial membranes, its analysis at the single-molecule level and its application in liposomal delivery. The whole protocol takes 4 days. Unlike mutagenesis, this method allows the introduction of custom-designed functional groups.

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References

  1. 1.

    & Semisynthetic proteins in mechanistic studies: using chemistry to go where nature can't. Curr. Opin. Chem. Biol. 10, 487–491 (2006).

  2. 2.

    , & Protein components for nanodevices. Curr. Opin. Chem. Biol. 9, 576–584 (2005).

  3. 3.

    & Functional engineered channels and pores. Mol. Membr. Biol. 21, 209–220 (2004).

  4. 4.

    , & Engineering light-gated ion channels. Biochemistry 45, 15129–15141 (2006).

  5. 5.

    et al. Reversibly caged glutamate: a photochromic agonist of ionotropic glutamate receptors. J. Am. Chem. Soc. 129, 260–261 (2007).

  6. 6.

    , , & Light-induced depolarization of neurons using a modified Shaker K(+) channel and a molecular photoswitch. J. Neurophysiol. 96, 2792–2796 (2006).

  7. 7.

    et al. Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat. Chem. Biol. 2, 47–52 (2006).

  8. 8.

    , & Photochemical tools for remote control of ion channels in excitable cells. Nat. Chem. Biol. 1, 360–365 (2005).

  9. 9.

    et al. Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 18, 1730–1737 (1999).

  10. 10.

    , , , & Release of thioredoxin via the mechanosensitive channel MscL during osmotic downshock of Escherichia coli cells. J. Biol. Chem. 273, 26670–26674 (1998).

  11. 11.

    , , & Elongation factor Tu and DnaK are transferred from the cytoplasm to the periplasm of Escherichia coli during osmotic downshock presumably via the mechanosensitive channel MscL. J. Bacteriol. 182, 248 (2000).

  12. 12.

    , , , & Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity. Biophys. J. 77, 1960–1972 (1999).

  13. 13.

    , & Chemically charging the pore constriction opens the mechanosensitive channel MscL. Biophys. J. 80, 2198–2206 (2001).

  14. 14.

    Liposomes: A Practical Approach, 105–161 (IRL Press, Oxford, 1990).

  15. 15.

    , , & A light-actuated nanovalve derived from a channel protein. Science 309, 755–759 (2005).

  16. 16.

    , , , , & Rationally designed chemical modulators convert a bacterial channel protein into a pH-sensory valve. Angew. Chem. Int. Ed. 45, 3126–3130 (2006).

  17. 17.

    et al. Alkali-metal cation recognition induced isomerization of spirobenzopyrans and spironapthoxazins possessing a crown ring as a recognition site—multifunctional artificial receptors. J. Org. Chem. 57, 5377–5383 (1992).

  18. 18.

    , & Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by Triton X-100, octyl glucoside, and sodium cholate. Biochemistry 27, 2668–2677 (1988).

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Acknowledgements

We thank the MscL team in Groningen for their support, Dr. G.T. Robillard for critical reading of the manuscript, the neurobiophysics group in University of Groningen for making available the patch-clamp facilities, C.M. Jeronimus-Stratingh and Annie van Dam for the ESI-MS analyses, the Netherlands Organization for Scientific Research (NWO-CW) (B.L.F.), the Materials Science Centre (MSC plus), University of Groningen (M.W. and B.L.F) and Nanoned, a national nanotechnology program coordinated by the Dutch Ministry of Economic Affairs (B.L.F. and A.K.) for financial support.

Author information

Author notes

    • Martin Walko

    Current address: Faculty of Sciences, Department of Organic Chemistry, Institute of Chemical Sciences, P.J. Safarik University, Moyzesova 11, 04001, Kosice, Slovakia.

    • Armağan Koçer
    •  & Martin Walko

    These authors contributed equally to this work.

Affiliations

  1. BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG Groningen, Netherlands.

    • Armağan Koçer
  2. Department of Organic and Molecular Inorganic Chemistry, Stratingh Institute and Material Science Centre, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

    • Martin Walko
    •  & Ben L Feringa

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Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Armağan Koçer or Ben L Feringa.

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DOI

https://doi.org/10.1038/nprot.2007.196

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