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Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

Author

Listed:
  • Moritz Pfreundschuh

    (Eidgenössische Technische Hochschule (ETH))

  • David Alsteens

    (Eidgenössische Technische Hochschule (ETH))

  • Ralph Wieneke

    (Institute of Biochemistry, Biocenter, Goethe-University Frankfurt)

  • Cheng Zhang

    (Stanford University School of Medicine
    University of Pittsburgh, School of Medicine)

  • Shaun R. Coughlin

    (Cardiovascular Research Institute, University of California San Francisco)

  • Robert Tampé

    (Institute of Biochemistry, Biocenter, Goethe-University Frankfurt)

  • Brian K. Kobilka

    (Stanford University School of Medicine)

  • Daniel J. Müller

    (Eidgenössische Technische Hochschule (ETH))

Abstract

A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

Suggested Citation

  • Moritz Pfreundschuh & David Alsteens & Ralph Wieneke & Cheng Zhang & Shaun R. Coughlin & Robert Tampé & Brian K. Kobilka & Daniel J. Müller, 2015. "Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM," Nature Communications, Nature, vol. 6(1), pages 1-7, December.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms9857
    DOI: 10.1038/ncomms9857
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    Cited by:

    1. Szymon P. Kordon & Kristina Cechova & Sumit J. Bandekar & Katherine Leon & Przemysław Dutka & Gracie Siffer & Anthony A. Kossiakoff & Reza Vafabakhsh & Demet Araç, 2024. "Conformational coupling between extracellular and transmembrane domains modulates holo-adhesion GPCR function," Nature Communications, Nature, vol. 15(1), pages 1-17, December.

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