title: Extraction of accurate biomolecular parameters from single-molecule force spectroscopy experiments.
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.
authors: Astbury Centre for Structural and Molecular Biology and School of Molecular and Cellular Biology, University of Leeds , Leeds, West Yorkshire, LS2 9JT, U.K.
authors: Oliver E
authors: OE
authors: Farrance
authors: Emanuele
authors: E
authors: Paci
authors: Sheena E
authors: SE
authors: Radford
authors: David J
authors: DJ
authors: Brockwell
mesh_terms: D001696:Biomechanical Phenomena; D055595:Mechanical Phenomena; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D011487:Protein Conformation; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins
keywords: atomic force microscopy (AFM); contour length; freely jointed chain (FJC); single-molecule force spectroscopy (SMFS); worm-like chain (WLC)