We have investigated coupling of lever arm rotation to the ATP binding and
hydrolysis steps for the myosin motor domain. In several current hypotheses
of the mechanism of force production by muscle, the primary mechanical fea
ture is the rotation of a lever arm that is a subdomain of the myosin motor
domain. In these models, the lever arm rotates while the myosin motor doma
in is free, and then reverses the rotation to produce force while it is bou
nd to actin. These mechanical steps are coupled to steps in the ATP hydroly
sis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation
to produce a more compact motor domain that has stored mechanical energy.
Our approach is to use transient electric birefringence techniques to measu
re changes in hydrodynamic size that result from lever arm rotation when va
rious Ligands are bound to isolated skeletal muscle myosin motor domain in
solution. Results for ATP and CTP, which do support force production by mus
cle fibers, are compared to those of ATP gamma S and GTP, which do not. Mea
surements are also made of conformational changes when the motor domain is
bound to NDP's and PPi in the absence and presence of the phosphate analogu
e orthovanadate, to determine the roles the nucleoside moieties of the nucl
eotides have on lever arm rotation. The results indicate that for the subst
rates investigated, rotation does not occur upon substrate binding, but is
coupled to the NTP hydrolysis step. The data are consistent with a model in
which only substrates that produce a motor domain-NDP-P-i complex as the s
teady-state intermediate make the motor domain more compact, and only those
substrates support force production.