title: Measuring Force-Induced Dissociation Kinetics of Protein Complexes Using Single-Molecule Atomic Force Microscopy.
abstract: Proteins respond to mechanical force by undergoing conformational changes and altering the kinetics of their interactions. However, the biophysical relationship between mechanical force and the lifetime of protein complexes is not completely understood. In this chapter, we provide a step-by-step tutorial on characterizing the force-dependent regulation of protein interactions using in vitro and in vivo single-molecule force clamp measurements with an atomic force microscope (AFM). While we focus on the force-induced dissociation of E-cadherins, a critical cell-cell adhesion protein, the approaches described here can be readily adapted to study other protein complexes. We begin this chapter by providing a brief overview of theoretical models that describe force-dependent kinetics of biomolecular interactions. Next, we present step-by-step methods for measuring the response of single receptor-ligand bonds to tensile force in vitro. Finally, we describe methods for quantifying the mechanical response of single protein complexes on the surface of living cells. We describe general protocols for conducting such measurements, including sample preparation, AFM force clamp measurements, and data analysis. We also highlight critical limitations in current technologies and discuss solutions to these challenges.
authors.affiliation: Iowa State University, Ames, IA, United States; Ames Laboratory, U.S. Department of Energy, Ames, IA, United States.
authors.forename: K
authors.initials: K
authors.lastname: Manibog
authors.affiliation: Iowa State University, Ames, IA, United States; Ames Laboratory, U.S. Department of Energy, Ames, IA, United States.
authors.forename: C F
authors.initials: CF
authors.lastname: Yen
authors.affiliation: Iowa State University, Ames, IA, United States; Ames Laboratory, U.S. Department of Energy, Ames, IA, United States. Electronic address: sivasank@iastate.edu.
authors.forename: S
authors.initials: S
authors.lastname: Sivasankar
mesh_terms: D015820:Cadherins / Q000737:chemistry* / Q000302:isolation & purification; D002448:Cell Adhesion / Q000235:genetics; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D015820:Cadherins; D046912:Multiprotein Complexes
keywords: Atomic force microscope; Catch bond, E-cadherin; Force clamp measurements; Ideal bond; Mechanobiology; Slip bond