title: How to Measure Load-Dependent Kinetics of Individual Motor Molecules Without a Force-Clamp.
abstract: Single-molecule force spectroscopy techniques, including optical trapping, magnetic trapping, and atomic force microscopy, have provided unprecedented opportunities to understand biological processes at the smallest biological length scales. For example, they have been used to elucidate the molecular basis of muscle contraction and intracellular cargo transport along cytoskeletal filamentous proteins. Optical trapping is among the most sophisticated single-molecule techniques. With exceptionally high spatial and temporal resolutions, it has been extensively utilized to understand biological functions at the single molecule level, such as conformational changes and force-generation of individual motor proteins or force-dependent kinetics in molecular interactions. Here, we describe a new method, "Harmonic Force Spectroscopy (HFS)." With a conventional dual-beam optical trap and a simple harmonic oscillation of the sample stage, HFS can measure the load-dependent kinetics of transient molecular interactions, such as a human β-cardiac myosin II interacting with an actin filament. We demonstrate that the ADP release rate of an individual human β-cardiac myosin II molecule depends exponentially on the applied load, which provides a clue to understanding the molecular mechanism behind the force-velocity curve of a contracting cardiac muscle. The experimental protocol and the data analysis are simple, fast, and efficient. This chapter provides a practical guide to the method: basic concepts, experimental setup, step-by-step experimental protocol, theory, data analysis, and results.
authors.affiliation: Department of Cellular and Molecular Pharmacology, The Howard Hughes Medical Institute, University of California, San Francisco, CA, United States.
authors.forename: J
authors.initials: J
authors.lastname: Sung
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: K I
authors.initials: KI
authors.lastname: Mortensen
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, United States.
authors.forename: J A
authors.initials: JA
authors.lastname: Spudich
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark. Electronic address: henrik.flyvbjerg@nanotech.dtu.dk.
authors.forename: H
authors.initials: H
authors.lastname: Flyvbjerg
mesh_terms: D008841:Actin Cytoskeleton; D024722:Cardiac Myosins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D052898:Optical Tweezers; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D024722:Cardiac Myosins
keywords: Cardiomyopathy; Force spectroscopy; Molecular motor; Myosin; Optical trap; Optical tweezer; Single molecule