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

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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Figure 1: Synthesis of iodoacetic acid 4,5-dimethoxy-2-nitrobenzyl ester (3) and the photochemical reaction of its MscL-bound form.
Figure 2: Synthesis of 3′,3′-dimethyl-1′-(2-iodoacetyloxyethyl) -6-nitrospirol[2H-1-benzopyran-2,2′-indoline] (6) and the photochemical reaction of its MscL-bound form.
Figure 3: A typical real-time fluorescent efflux assay.
Figure 4: Electrophysiological analysis of reversible functioning of modified MscL.

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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.

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Correspondence to Armağan Koçer or Ben L Feringa.

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Koçer, A., Walko, M. & Feringa, B. Synthesis and utilization of reversible and irreversible light-activated nanovalves derived from the channel protein MscL. Nat Protoc 2, 1426–1437 (2007). https://doi.org/10.1038/nprot.2007.196

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