Muscle myosin performance measured with a synthetic nanomachine reveals a class-specific Ca2+-sensitivity of the frog myosin II isoform

A 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.

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 (F0, 15.25 pN) is increased by 50% (P = 0.004) with respect to that in Ca2+-free solution, the maximum shortening velocity (V0, 4.6 ?m s-1) is reduced by 27% (P = 0.46) and the maximum power (Pmax, 7.6 aW) is increased by 21% (P = 0.17). V0 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 (Vf). The rate of ATP-hydrolysis in solution (?) exhibits a similar calcium dependence. Ca2+ titration curves for Vf and ? give Kd values of ~30 ?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.