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Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Pascal Poc, View ORCID ProfileVanessa A. Gutzeit, View ORCID ProfileJulia Ast, Joon Lee, Ben J. Jones, View ORCID ProfileElisa D’Este, Bettina Mathes, View ORCID ProfileDavid J. Hodson, Joshua Levitz, View ORCID ProfileJohannes Broichhagen
doi: https://doi.org/10.1101/2020.01.29.924829
Pascal Poc
1Max Planck Institute for Medical Research, Department of Chemical Biology, Jahnstr. 29, 69120 Heidelberg, Germany
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Vanessa A. Gutzeit
2Neuroscience Graduate Program, Weill Cornell Medicine, New York, NY 10065
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  • ORCID record for Vanessa A. Gutzeit
Julia Ast
3Institute of Metabolism and Systems Research (IMSR), and Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK
4Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK
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Joon Lee
5Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065
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Ben J. Jones
6Section of Investigative Medicine, Imperial College London, London W12 0NN, United Kingdom
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Elisa D’Este
7Optical Microscopy Facility, Max Planck Institute for Medical Research, Heidelberg, Germany
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  • ORCID record for Elisa D’Este
Bettina Mathes
1Max Planck Institute for Medical Research, Department of Chemical Biology, Jahnstr. 29, 69120 Heidelberg, Germany
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David J. Hodson
3Institute of Metabolism and Systems Research (IMSR), and Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK
4Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK
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  • ORCID record for David J. Hodson
  • For correspondence: d.hodson@bham.ac.uk jtl2003@med.cornell.edu johannes.broichhagen@mr.mpg.de
Joshua Levitz
5Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065
8Tri-Institutional PhD Program in Chemical Biology, New York, NY 10065
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  • For correspondence: d.hodson@bham.ac.uk jtl2003@med.cornell.edu johannes.broichhagen@mr.mpg.de
Johannes Broichhagen
1Max Planck Institute for Medical Research, Department of Chemical Biology, Jahnstr. 29, 69120 Heidelberg, Germany
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  • ORCID record for Johannes Broichhagen
  • For correspondence: d.hodson@bham.ac.uk jtl2003@med.cornell.edu johannes.broichhagen@mr.mpg.de
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Abstract

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

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Posted January 30, 2020.
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Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates
Pascal Poc, Vanessa A. Gutzeit, Julia Ast, Joon Lee, Ben J. Jones, Elisa D’Este, Bettina Mathes, David J. Hodson, Joshua Levitz, Johannes Broichhagen
bioRxiv 2020.01.29.924829; doi: https://doi.org/10.1101/2020.01.29.924829
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Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates
Pascal Poc, Vanessa A. Gutzeit, Julia Ast, Joon Lee, Ben J. Jones, Elisa D’Este, Bettina Mathes, David J. Hodson, Joshua Levitz, Johannes Broichhagen
bioRxiv 2020.01.29.924829; doi: https://doi.org/10.1101/2020.01.29.924829

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