Mechanokinetic properties of an ensemble of myosin II molecular motors purified from skeletal muscle and reassembled in a sarcomere-like nanomachine

This Thesis focuses on the investigation of the molecular mechanism of contraction in striated muscle, with the aim of providing a theoretical framework that could contribute to the definition of the mechano-kinetic parameters underlying the performance emerging from the array arrangement of myosin motors in the half-sarcomere, the functional unit of the striated muscle. To this end, the mechanical output of a synthetic sarcomere-like nanomachine, in which a small ensemble of myosin molecules purified from the fast and slow skeletal muscles of the rabbit interacts with an actin filament, is recorded with a Dual Laser Optical Tweezers and fed to a stochastic model which provides a self–consistent estimate of all the mechanokinetic parameters of the motor ensemble. Through a process of reverse engineering, it was possible to recover isoform-dependent single motor properties from the analysis of the dynamics of the force of the whole ensemble. In fact, data fitting of the time series of the experimental force of the ensemble provided a self-consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin-attached motors, and the rate of transition through the attachment-detachment cycle. Results show that, in isometric conditions and with respect to the slow myosin isoform, the fast myosin isoform shows more than twice the force of a single motor, a lower fraction of attached motors in the ensemble, and more than twice the rate of flux through the acto-myosin interaction cycle. The achievements in this Thesis set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules purified from animal models or human biopsies and pave the way for defining the performance of unknown isoforms and mutant and engineered myosin motors.