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Reversible site-selective labeling of membrane proteins in live cells

Nature Biotechnology volume 22, pages 440–444 (2004)Cite this article

Abstract

Chemical and biological labeling is fundamental for the elucidation of the function of proteins within biochemical cellular networks. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution1,2,3. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest4. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates5 to a representative ligand-gated ion channel and G protein–coupled receptor, each containing a polyhistidine sequence. We investigated the ionotropic 5HT3 serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.

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Figure 1: Labeling GFP-His6 by NTA probes.
Figure 2: Structure of 5HT3R in live cells investigated by FRET using NTA probes.
Figure 3: In vivo labeling of ionotropic and G protein–coupled receptors.

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Acknowledgements

We are grateful to several colleagues: Horst Blasey for the stable cell line expressing 5HT3R-C-His6; Bruno Meyer for construction and functional expression of GFP-NK1-His10; David Lovinger, Horst Pick, Erwin Ilegems, CĂ©dric Deluz and Kenneth Lundstrom for plasmids encoding 5HT3R, 5HT3R-Loop-His6, 5HT3R-N-His6, 5HT3R-C-His10 and NK1R; Andreas Heusler for synthesizing NTA-Rho; Annmarie Surprenant and Christoph Schreiter for the electrophysiological characterization of the oligohistidine-tagged 5HT3Rs; Martha Liley and Daniel Abankwa for carefully reading the manuscript. This work was financially supported by the Swiss National Science Foundation.

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Author notes

  1. Emmanuel G Guignet and Ruud Hovius: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Physical Chemistry of Polymers and Membranes, Institute of Biomolecular Sciences, Swiss Federal Institute of Technology, Lausanne, 1015, Switzerland

    Emmanuel G Guignet, Ruud Hovius & Horst Vogel

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  1. Emmanuel G Guignet
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Correspondence to Horst Vogel.

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Guignet, E., Hovius, R. & Vogel, H. Reversible site-selective labeling of membrane proteins in live cells. Nat Biotechnol 22, 440–444 (2004). https://doi.org/10.1038/nbt954

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