Single-Molecule Force Spectroscopy: A Method for Quantitative Analysis of Ligand–Receptor Interactions

Abstract

The quantitative analysis of molecular interactions is of high interest in medical research. Most methods for the investigation of ligand-receptor complexes deal with huge ensembles of biomolecules, but often neglect interactions with low affinity or small subpopulations with different binding properties. Single-molecule force spectroscopy offers fascinating possibilities for the quantitative analysis of ligand-receptor interactions in a wide affinity range and the sensitivity to detect point mutations. Furthermore, this technique allows one to address questions about the related binding energy landscape. In this article, we introduce single-molecule force spectroscopy with a focus on novel developments in both data analysis and theoretical models for the technique. We also demonstrate two examples of the capabilities of this method.

Atomic force microscopy: A multifaceted tool to study membrane proteins and their interactions with ligands

“…Using the value derived for k 0 , the free energy of activation for protein-ligand dissociation, ΔG ‡ , can be estimated from the Arrhenius equation [61]. Updated models are also actively in development to improve and overcome some of the limitations of the Bell-Evans model to describe DFS data (e.g., [62–64]). A non-linear relationship between force and logarithm of the loading rate, where there are multiple linear regimes (Fig.…”

“…The method has high sensitivity and has been used to investigate protein-ligand interactions with equilibrium dissociation constants ranging from fM to µM concentrations [64]. Dissociation rate constants can be determined without considering the complications that exist in more traditional assays due to the reassociation of the protein-ligand complex [64].…”

“…The method has high sensitivity and has been used to investigate protein-ligand interactions with equilibrium dissociation constants ranging from fM to µM concentrations [64]. Dissociation rate constants can be determined without considering the complications that exist in more traditional assays due to the reassociation of the protein-ligand complex [64]. Association rate constants can also be computed by determining the probability of interactions between the ligand and protein by varying the contact time between the functionalized AFM probe and the membrane surface [67].…”

Mechanochemitry: A Molecular Biomechanics View of Mechanosensing

“…It follows from the Bell equation that when a ramp force is applied to rupture a molecular bond, the most probable rupture force, identified from the peak of the histogram of a large population of rupture forces measured under identical conditions, depends linearly on the logarithm of the ramp rate. Based on this prediction, the method of dynamic force spectroscopy (DFS) was developed 12,26,27 , leading to a rapid growth of research that analyzed the force-dependent dissociation of a large number of molecular interactions 28,29 . The theoretical basis of DFS has also been further extended to include other effects such as those of the elastic potential of the molecular complex and the force probe 30,31 .…”

Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices

“…Atomic force microscopy (AFM) [5], primarily known as a high resolution imaging technique, can be utilized for precise force measurements with pN resolution [6–11]. In single cell force spectroscopy (SCFS) [2,12–22], AFM is applied to manipulate single living cells and quantify interaction forces between the cells and substrates or other cells.…”