Hidden Multiple Bond Effects in Dynamic Force Spectroscopy

Abstract

In dynamic force spectroscopy, a (bio-)molecular complex is subjected to a steadily increasing force until the chemical bond breaks. Repeating the same experiment many times results in a broad distribution of rupture forces, whose quantitative interpretation represents a formidable theoretical challenge. In this study we address the situation that more than a single molecular bond is involved in one experimental run, giving rise to multiple rupture events that are even more difficult to analyze and thus are usually eliminated as far as possible from the further evaluation of the experimental data. We develop and numerically solve a detailed model of a complete dynamic force spectroscopy experiment including a possible clustering of molecules on the substrate surface, the formation of bonds, their dissociation under load, and the postprocessing of the force extension curves. We show that the data, remaining after elimination of obvious multiple rupture events, may still contain a considerable number of hidden multiple bonds, which are experimentally indistinguishable from true single bonds, but which have considerable effects on the resulting rupture force statistics and its consistent theoretical interpretation.

Calcium increases titin N2A binding to F-actin and regulated thin filaments

“…The most likely cause of this wide force distribution is random variation in the interaction due to stochastic thermal fluctuations and variable loading directions 42 , 43 . Other possible explanations include multiple N2A - F-actin interactions 44 or multiple F-actin binding sites within each N2A molecule 27 , but both are unlikely. It is unlikely that the wide distribution is due to multiple N2A molecules interacting with F-actin because only force curves demonstrating single interaction events were included in the analysis.…”

How Do We Know when Single-Molecule Force Spectroscopy Really Tests Single Bonds?

“…A better approach is to filter out force cycles with multiple rupture events (3,32,33). However, this assumes that two rupture events will be spatially separated, which often fails when the molecules are short or when adhesion probability is high (34,35). Nevertheless, this simple filtering method can improve the accuracy of experiments obtained with low adhesion probabilities, especially if the fraction of data filtered out correspond with the statistical predictions.…”

“…Some studies have analyzed this force data by only considering the last rupture event in the force cycle (59,60), but this again assumes that two rupture events must be spatially separated. This assumption fails especially when there are many rupture events, resulting in hidden multiple interactions (34,35). Another shortcoming is that these methods assume prior knowledge about the appearance of single molecule data and have not been shown to be reliable for the complex energy landscapes described above.…”

The biophysics of bacterial infections: Adhesion events in the light of force spectroscopy

“…Although the Bell-Evans effect has been and still is the backbone of force spectroscopy data analysis, biological bonds are complex systems. In this context, many biological experimental data do not fit within the theoretical expectations, mainly because there are multiple molecule events, instead of truly single molecule data ( Chang et al, 2015 , Fuhrmann, 2015 , Fuhrmann et al, 2012 , Getfert and Reimann, 2012 , Raible et al, 2006 ). This gives rise to another important aspect that is the number of bonds formed at each tip-sample contact, i.e , how to try to guarantee specific single bond events.…”