Abstract
Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using ‘harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.
Highlights
Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction
Several investigations of non-human skeletal or cardiac low-duty-ratio myosins have used either an ensemble of molecules in an in vitro motility assay at a saturating adenosine triphosphate (ATP) concentration (Z2 mM)[8,9] or a mini-ensemble in an optical trap assay at sub-saturating ATP concentrations[10,11] to examine the collective load-dependent behaviour of myosin motors
Optical traps are used in a dual-beam filament–dumbbell configuration to probe a nonprocessively interacting motor anchored to a surface
Summary
Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. There have been other approaches for increased detection speed and sensitivity by oscillating either the trap or the stage[25,26,27,28,29], and, recently, an ultrafast force-clamp optical trap with sub-millisecond temporal resolution was developed by Capitanio et al.[29] To achieve that, the actin dumbbell was preloaded by oscillating it at a constant speed in a triangular wave-form, and binding events were detected as deviations in position of the bead from the trap These clamped-force configurations all require complex instrumentation and careful operation for fast and robust feedback control, which might not be adaptable or extendable to a variety of systems. This simplicity makes ‘harmonic force spectroscopy’ an easy assay with improved sampling efficiency as compared with existing methods, fast enough to achieve single-molecule results from single-molecule experiments
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