Mouse Myosinviia is a Monomeric, “slow” Motor with an Intermediate Duty Ratio

Abstract

Myosin VIIa is an unconventional myosin which is important in visual and hearing processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing all 5IQ motifs and the SAH domain (myosinVIIa-S1-SAH). The construct is single-headed with a molecular weight of ∼ 130 kDA determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by atomic force microscopy. The relatively high basal steady-state rate of 0.18±0.05 s−1 is 3-fold activated by actin with a Vmax of 0.6± 0.02 s−1 and a KATPase of 11.5± 2.9 μM. The maximal rate of phosphate dissociation from actomyosinVIIA-ADP-Pi complex measured by the fluorescently labeled phosphate-binding protein could not be obtained because of the weak binding of the myosinVIIA-ADP-Pi complex to actin. A rate of 2.6s−1 was measured in the presence of 45uM actin. Double mixing stopped-flow experiments measure two rates 4.0 s−1 and 0.9 s−1 of ADP dissociation from the actomyosin-ADP complex and a rate of 2.0 s−1 from myosin-ADP. ATP binds to myosinVIIa with a rate constant of 3.2 uM−1s−1. ATP hydrolysis measured by quenched flow gave a rate of 10 s−1, which correlated well with the maximal rate of 13 s−1 measured by tryptophan fluorescence. The equilibrium constant of the hydrolysis (KH) is ∼ 1. These data show that mouse myosin VIIa-S1-SAH is a “slow” monomeric, molecular motor with an intermediate duty ratio of >0.4. Therefore for myosin VIIa to reach a high effective duty ratio several myosin molecules are brought together and localized to the actin bundles in the stereocilia participating in the lateral connections along with the interacting proteins such as vezatin and cadherin.This work was supported by NIH/DC 009335 to EF and NIH/EB00209 to HDW.