This thesis reports the realization and first application of a synthetic nanomachine, able to reproduce in vitro the performance emerging from the array arrangement of myosin II motors in the sarcomere of the striated muscle. The nanomachine consists of an ensemble of less than ten myosin dimers from fast skeletal muscle disposed on a functionalized support carried by a piezoelectric nanopositioner and brought to interact with an actin filament attached with the correct polarity via gelsolin to a bead (Bead Tailed Actin, BTA) trapped into the focus of a Dual Laser Optical Tweezers (DLOT). In solution with [ATP] = 2 mM the nanomachine is able to produce steady force and shortening, delivering a maximum power of 5 aW. The nanomachine performances are interpreted with a kinetic model based on mechanics and energetics of fast skeletal muscle. In this way it is possible to define the minimal conditions that allow an actomyosin system in vitro to produce force and power with the efficiency of the striated muscle, in the absence of the confusing contribution of the other sarcomeric proteins. In turn, since the system is assembled one piece at a time, it allows different degrees of reconstitution of the sarcomeric assembly. Therefore it will be possible to characterize the function of native and engineered contractile, regulatory and accessory proteins. For future investigations on the Ca2+-dependent thin filament activation, the preparation of BTA has been implemented using a Ca2+-independent gelsolin fragment and the procedure for thin filament reconstitution has been established during my visit to the Institute for Biophysical Chemistry, MHH, Germany.
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|Titolo:||The power output of a myosin II-based nanomachine mimicking the striated muscle|
|Appare nelle tipologie:||8.1 Tesi Dottorato|