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The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, the generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine made of an ensemble of eight heavy-meromyosin (HMM) fragments of myosin from rabbit psoas muscle, carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca 2+ -regulated actin binding protein) to a bead trapped by dual laser optical tweezers. However, the application of the original version of the nanomachine to investigate the Ca 2+ -dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them one at a time, was prevented by the impossibility to preserve [Ca 2+ ] as a free parameter. Here, the nanomachine is implemented by assembling the bead-attached actin filament with the Ca 2+ -insensitive gelsolin fragment TL40. The performance of the nanomachine is determined both in the absence and in the presence of Ca 2+ (0.1 mM, the concentration required for actin attachment to the bead with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca 2+ ], opening the possibility for future studies of the Ca 2+ -dependent function/dysfunction of regulatory and cytoskeletal proteins.

A Myosin II-Based Nanomachine Devised for the Study of Ca2+-Dependent Mechanisms of Muscle Regulation