Abstract
The atomic force microscope (AFM) is able to manipulate biomolecules and their complexes with exquisite force sensitivity and distance resolution. This capability, complemented by theoretical models, has greatly improved our understanding of the determinants of mechanical strength in proteins and revealed the diverse effects of directional forces on the energy landscape of biomolecules. In unbinding experiments, the interacting partners are usually immobilized on their respective substrates via extensible linkers. These linkers affect both the force and contour length (Lc) of the complex at rupture. Surprisingly, while the former effect is well understood, the latter is largely neglected, leading to incorrect estimations of Lc, a parameter that is often used as evidence for the detection of specific interactions and remodeling events and for the inference of interaction regions. To address this problem, a model that predicts contour length measurements from single-molecule forced-dissociation experiments is presented that considers attachment position on the AFM tip, geometric effects, and polymer dynamics of the linkers. Modeled data are compared with measured contour length distributions from several different experimental systems, revealing that current methods underestimate contour lengths. The model enables nonspecific interactions to be identified unequivocally, allows accurate determination of Lc, and, by comparing experimental and modeled distributions, enables partial unfolding events before rupture to be identified unequivocally.
Highlights
The atomic force microscope (AFM) is able to manipulate biomolecules and their complexes with exquisite force sensitivity and distance resolution
Single-molecule force spectroscopy (SMFS) techniques are increasingly being applied in biology to understand biomolecular interactions and protein dissociation under the application of force.1À4 Usually employed in nonequilibrium conditions, SMFS offers unique insights into the kinetics of bond dissociation of inter- and intramolecular interactions under applied force, allowing exploration of regions of the underlying free energy landscape that are inaccessible to traditional ensemble methods.[5]
The contour length that is measured in an AFM-based SMFS experiment corresponds to the separation between the two surfaces when the linkers, tethered by complex formation, are fully extended (Figure 1)
Summary
The atomic force microscope (AFM) is able to manipulate biomolecules and their complexes with exquisite force sensitivity and distance resolution. While the former effect is well understood, the latter is largely neglected, leading to incorrect estimations of Lc, a parameter that is often used as evidence for the detection of specific interactions and remodeling events and for the inference of interaction regions To address this problem, a model that predicts contour length measurements from single-molecule forced-dissociation experiments is presented that considers attachment position on the AFM tip, geometric effects, and polymer dynamics of the linkers. In mechanical unfolding experiments, where a single protein is extended between the tip and AFM substrate, the protein is usually immobilized directly to the mica,[24] glass,25À27 or gold28À30 substrate and attached to the tip via nonspecific adsorption As the latter requires the application of pressure onto the polypeptide by the tip, immobilization presumably occurs close to the apex, and the contour length that is measured (found by fitting of an appropriate model to forceÀdistance curves) is usually close to the end-toend length of the immobilized protein (Figure 1b), irrespective of differences in pulling geometry.[31]
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