Force Transducer–Dependent Mechanics in Biomolecular Complexes: Implications for Dynamic Force Spectroscopy Models

Abstract

Understanding the mechanical behavior of bacterial adhesion complexes, such as the interaction between the adhesin SD-repeat protein G (SdrG) from Staphylococcus epidermidis and the human fibrinogen β-chain (Fgβ), is essential for developing strategies to combat infections. Over the past decades, theoretical frameworks have been developed to interpret the force-dependent behavior of biomolecular systems undergoing transitions such as unfolding or unbinding. Among these, the Bell–Evans (BE) and Dudko–Hummer–Szabo (DHS) models are widely used to analyze data from single-molecule force spectroscopy (SMFS) experiments, including both force-clamp and force-ramp protocols. The DHS model is particularly important because it provides a unified framework for interpreting results from both atomic force microscopy (AFM)-based experiments and steered molecular dynamics (SMD) simulations, which operate across distinct loading-rate regimes. However, recent work from our group suggests that the stiffness of the force transducer introduces an additional effect on the measured force response, particularly for conditions outside the soft-spring regime in which the standard DHS implementation is expected to hold; while theoretical treatments addressing finite transducer stiffness and rapid loading already exist in the literature, their implications have not previously been characterized systematically for a biologically relevant, irreversibly dissociating adhesion complex. In this work, we perform a systematic series of SMD-based SMFS simulations to investigate the role of force transducer stiffness in the dissociation of the SdrG:Fgβ complex within the DHS framework. By varying the spring constant over a range spanning three orders of magnitude (0.1 to 100.0 kcal mol−1 Å−2) and analyzing rupture forces within equivalent loading-rate ranges, we observe a systematic increase in the instantaneous peak rupture force with increasing stiffness; notably, this apparent stiffness dependence is substantially reduced when rupture force is instead defined from a linear fit to the averaged force ramp, indicating that the effect is sensitive to how rupture force is operationally defined. Under the peak-force definition, this effect leads to significant variations in the DHS parameters extracted by fitting (the activation free-energy barrier ΔG‡, the distance to the transition state x‡, and the zero-force dissociation rate k0); given their strong mutual correlation and the limited identifiability of k0 in particular, we treat these as effective fit parameters rather than as uniquely determined, intrinsic properties of the molecular system. Despite these differences in the measured force response, analysis of heavy-atom contacts and consensus dissociation pathways reveals no large-scale, systematic structural variation across stiffness conditions, within the resolution of these analyses. This apparent stiffness dependence largely collapses when rupture force is instead defined via a linear fit to the averaged force ramp rather than the instantaneous peak, and it is not accompanied by any large-scale, systematic change in the dissociation pathway detected by contact or consensus-path analysis, indicating that the operational definition of rupture force, rather than a genuine shift in the underlying molecular mechanism, is the primary driver of the observed differences. Our results indicate that the standard DHS implementation does not provide stiffness-independent parameters across the full range examined here, particularly outside its soft-spring applicability regime, leading to potential inconsistencies in the interpretation of SMFS data when stiffness and the definition of rupture force are not explicitly accounted for. These findings highlight the need for further investigation into the origin of this sensitivity, and for care in reporting both transducer stiffness and rupture-force definition when comparing SMFS measurements across studies.