Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultra Force-Clamp Spectroscopy

Year: 2021

Authors: Gardini L., Kashchuk AV., Pavone FS., Capitanio M.

Autors Affiliation: CNR, Natl Inst Opt, Florence, Italy; European Lab Nonlinear Spect, LENS, Sesto Fiorentino, Italy; Univ Florence, Phys Dept, Florence, Italy.

Abstract: Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force. By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Journal/Review: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS

Volume: (173)      Pages from: e62388-1  to: e62388-14

More Information: This work was supported by the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 871124 Laserlab-Europe, by the Italian Ministry of University and Research (FIRB Futuro in Ricerca 2013 Grant No. RBFR13V4M2), and by Ente Cassa di Risparmio di Firenze. A.V. Kashchuk was supported by Human Frontier Science Program Cross-Disciplinary Fellowship LT008/2020-C.
KeyWords: Optical Tweezers
DOI: 10.3791/62388

ImpactFactor: 1.424

Connecting to view paper tab on IsiWeb: Click here