title: Detecting Ligand-Binding Events and Free Energy Landscape while Imaging Membrane Receptors at Subnanometer Resolution.
abstract: Force-distance curve-based atomic force microscopy has emerged into a sophisticated technique for imaging cellular membranes and for detecting specific ligand-binding events of native membrane receptors. However, so far the resolution achieved has been insufficient to structurally map ligand-binding sites onto membrane proteins. Here, we introduce experimental and theoretical approaches for overcoming this limitation. To establish a structurally and functionally well-defined reference sample, we engineer a ligand-binding site to the light-driven proton pump bacteriorhodopsin of purple membrane. Functionalizing the AFM stylus with an appropriate linker-system tethering the ligand and optimizing the AFM conditions allows for imaging the engineered bacteriorhodopsin at subnanometer resolution while structurally mapping the specific ligand-receptor binding events. Improved data analysis allows reconstructing the ligand-binding free energy landscape from the experimental data, thus providing thermodynamic and kinetic insight into the ligand-binding process. The nanoscopic method introduced is generally applicable for imaging receptors in native membranes at subnanometer resolution and for systematically mapping and quantifying the free energy landscape of ligand binding.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich , 4058 Basel, Switzerland.
authors.forename: Moritz
authors.initials: M
authors.lastname: Pfreundschuh
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern , 3012 Bern, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Harder
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern , 3012 Bern, Switzerland.
authors.forename: Zöhre
authors.initials: Z
authors.lastname: Ucurum
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern , 3012 Bern, Switzerland.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich , 4058 Basel, Switzerland.
authors.forename: Daniel J
authors.identifier: 0000-0003-3075-0665
authors.initials: DJ
authors.lastname: Müller
keywords: Ligand−receptor binding; free energy landscape; membrane proteins; multiparametric imaging; single-molecule force spectroscopy; single-molecule imaging