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.

Nanomechanical mapping of soft materials with the atomic force microscope: methods, theory and applications

“…The combination of FV and single-molecule force spectroscopy 2,117 might lead to a new nanomechanical force spectroscopy method. Initial experimental results were promising 118,119 but they involved a very small region of the sample mapped by FV. It is not straightforward to envision how the requirements of singlemolecule force spectroscopy, namely, the acquisition of multiple FDCs at different loading rates 120 on the same position of the sample might be compatible with a fast acquisition of FV maps.…”

Binding Strength of Gram-Positive Bacterial Adhesins

“…From the FD plot, the magnitude of the binding strength (or adhesion force) in piconewtons (pN) can be assessed, together with other biophysical parameters. By varying the pulling speed, dynamic force spectroscopy data are generated (Pfreundschuh et al, 2017). Modeling such data with appropriate theories provides quantitative information on thermodynamic and kinetic parameters of the single-molecule interaction.…”

Dynamics of breaking intermolecular bonds in high-speed force spectroscopy

“…The above prediction has been verified experimentally in many SMFS experiments performed at low to moderate loading rates (10 2 to 10 5 pN/s). 4,14,18,[22][23][24][25][26][27][28][29][30] In those experiments, the rupture force has been postulated to coincide with the measured force F rup ≡F m =kz rup . The measured force is determined from the cantilever deflection at the rupture distance, z rup .…”