In vitro study of the mechano–kinetic properties of muscle myosin

The aim of the project is to build a sarcomere-like machine consisting of a syntheticarray of myosin motor surface brought to interact with an actin filament. The ensemble of myosin motors will provide the condition for generating steady force and shortening by cyclic interactions with the actin filament.The mechanical outputs of the machine will be measured by means of a Dual Laser Optical Tweezers apparatus (DLOT, range 0.5-200 pN force,1-10000 nm displacement), able to control either the movement or the force of the biomachine.The motor protein chosen is theHMM fraction of myosin II from frog and rabbit skeletal muscle.Mechanical measurements are done with a simplified version of the machine, in which the number of the motors interacting with the actin filament is limited by using as a support the TMCS (Trimethylchlorosilane) coated flat tip of an etched optical fibre (diameter 3-8 µm). The position of the fibre is controlled with a three-axes piezo-stage acting as a length transducer. The correct polarity of the actin filament (5-15 µm length, purified from rabbit psoas) is controlled by attaching its barbed end to a bead that is trapped into the focus of the DLOT acting as a force transducer. The experiments (temperature 24°C, ionic strength 60 mM) include measurements of the rupture force and lifetime of the actin-HMM bound in rigor (ATP-free solution) and of the active force developed by the biomachine in 2 mMMgATP.The rupture force of the rigor bond is 12.85 ± 0.35 pN. The bond lifetime under a load of 8 pN has a bi-exponential distribution and the time constant of the major, faster component is 1 s. When the ensemble of myosin motors is perfused with2 mMMgATPforce develops to a steady value of 50 pN in 2 s. Taking into account the compliance of the trap (7 nm/pN), the sliding of the actin filament during force development amounts to 350 nm, which is accounted for by ATP driven cycles of detachment/reattachment of individual myosin motors.Supported by IIT(Genova) and PRIN 2011 (MIUR, Italy)