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

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 μm 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 (φ) exhibits a similar calcium dependence. Ca2+ titration curves for Vf and φ give Kd values of ∼30 μm. 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.