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

Abstract

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. 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 2mm ATP is affected by Ca2+ : in 0.1mm Ca2+ , the isometric steady force (F0 , 15.25pN) is increased by 50% (P=0.004) with respect to that in Ca2+ -free solution, the maximum shortening velocity (V0 , 4.6μm s-1 ) is reduced by 27% (P=0.46) and the maximum power (Pmax , 7.6aW) 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 ∼30μm. 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.