Muscle Myosin Performance Measured With A Synthetic Nanomachine Reveals A Class-Specific Ca2+-Sensitivity Of The Frog Myosin Ii Isoform

Key pointsA nanomachine made of an ensemble of seven heavy-meromyosin (HMM) fragments of muscle myosin interacting with an actin filament is able to mimic the half-sarcomere generating steady force and constant-velocity shortening.To preserve Ca2+ as a free parameter, the Ca2+-insensitive gelsolin fragment TL40 is used to attach the correctly oriented actin filament to the laser-trapped bead acting as a force transducer.The new method reveals that the performance of the nanomachine powered by myosin from frog hind-limb muscles depends on [Ca2+], an effect mediated by a Ca2+-binding site in the regulatory light chain of HMM. The Ca2+-sensitivity is class-specific because the performance of the nanomachine powered by mammalian skeletal muscle myosin is Ca2+ independent.A model simulation is able to interface the nanomachine performance with that of the muscle of origin and provides a molecular explanation of the functional diversity of muscles with different orthologue isoforms of myosin.An ensemble of seven heavy-meromyosin (HMM) fragments of myosin-II purified from the hindlimb muscles of the frog (Rana esculenta) is used to drive a synthetic nanomachine that pulls an actin filament in the absence of confounding effects of other sarcomeric proteins. In the present version of the nanomachine the +end of the actin filament is attached to the laser trapped bead via the Ca2+-insensitive gelsolin fragment TL40, making [Ca2+] a free parameter. Frog myosin performance in 2 mm ATP is affected by Ca2+: in 0.1 mm Ca2+, the isometric steady force (F-0, 15.25 pN) is increased by 50% (P = 0.004) with respect to that in Ca2+-free solution, the maximum shortening velocity (V-0, 4.6 mu m s(-1)) is reduced by 27% (P = 0.46) and the maximum power (P-max, 7.6 aW) is increased by 21% (P = 0.17). V-0 reduction is not significant for the paucity of data at low force, although it is solidified by a similar decrease (33%, P < 0.0001) in the velocity of actin sliding as indicated by an in vitro motility assay (V-f). The rate of ATP-hydrolysis in solution (phi) exhibits a similar calcium dependence. Ca2+ titration curves for V-f and phi give K-d values of similar to 30 mu m. All the above mechanical and kinetic parameters are independent of Ca2+ when HMM from rabbit psoas myosin is used, indicating that the Ca2+-sensitivity is a class-specific property of muscle myosin. A unique multiscale model allows interfacing of the nanomachine performance to that of the muscle of origin and identifies the kinetic steps responsible for the Ca2+-sensitivity of frog myosin.