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

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

  • Nat Commun. 2015 Nov 12;6:8857. doi: 10.1038/ncomms9857.
Moritz Pfreundschuh 1,  David Alsteens 1,  Ralph Wieneke 2,  Cheng Zhang 3 4,  Shaun R Coughlin 5,  Robert Tampé 2,  Brian K Kobilka 3,  Daniel J Müller 1
Affiliations

Affiliations

  • 1 Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH), Mattenstrasse 26, 4058 Basel, Switzerland.
  • 2 Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, 60438 Frankfurt/Main, Germany.
  • 3 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA.
  • 4 Department of Pharmacology and Chemical Biology, University of Pittsburgh, School of Medicine, E1358 Thomas E. Starzl BST, Pittsburgh, Pennsylvania 15261, USA.
  • 5 Cardiovascular Research Institute, University of California San Francisco, 555 Mission Bay Boulevard South, S452P, San Francisco, California 94158, USA.
PMID: 26561004 DOI: 10.1038/ncomms9857
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.

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