The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actinbased motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin (20-fold) with K actin of 112 M and V max of 0.34 s ؊1 , indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity (0.11 ؎ 0.05 m/s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C-terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin-based motor protein having a protein kinase activity.Myosin III is a member of the myosin superfamily, which consists of at least 18 classes (1-5). The class III myosin represents the most divergent member of the myosin superfamily. The motor domain of myosin III shows ϳ24 -27% sequence identity to myosin Is and ϳ22-26% sequence identity to myosin-IIs (6). Of particular interest is that myosin III has an amino terminus domain that resembles to protein kinases. This domain contains the characteristic motifs for protein kinases such as a glycine-rich loop, an invariable Lys residue required for ATP binding, and a catalytic loop. Given its high degree of divergence, if myosin III evolved at the same rate as other myosins, the myosin III lineage would predate the divergence of yeast. Class III myosin was originally found in Drosophila photoreceptor cells and subsequently found in vertebrates including human myosin III (7,8).The function of Myosin III is best studied in Drosophila photoreceptor cells. Each photoreceptor cell has a specialized organelle consisting of a stack of microvilli known as a rhabdomere. The phototransduction machinery is localized in the rhabdomere (9). Drosophila photoreceptors undergo a prolonged depolarization afterpotential that persists after cessation of the light stimulus. Prolonged depolarization afterpotential results from the stable conversion of rhodopsin to the light-activated form, metarhodopsin, in response to blue light. During a prolonged depolarization afterpotential, photoreceptor cells become refractory to subsequent prolonged depolarization afterpotential-inducing stimuli and are inactivated. Mutants that are defective for both inactivation and the prolonged depolarization afterpotential are known as neither inactivation nor afterpotential (nina) mutants (10, 11), and the myosin III was identified (12) as one of eight nina complementation gr...

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Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin to reversibly regulate the ATPase activity of kinesin by photoirradiation. We prepared five kinesin motor domain mutants, A247C, L249C, A252C, G272C and S275C, which contained a single reactive cysteine residue in loops L11 and L12. These loops are considered to be key regions for the functioning of kinesin as a motor protein. PAM was stoichiometrically incorporated into the cysteine residues in the loops of the mutants. The PAM-modified S275C mutant exhibited reversible alterations in ATPase activity accompanied by cis-trans isomerization upon UV and VIS light irradiation. The ATPase activity exhibited by the cis-isomer of the PAM bound to the mutant was two times higher than that of the trans-isomer. Further, the PAM-modified L249C mutant exhibited reversible alterations in ATPase activity on UV-VIS light irradiation but exhibited the opposite effect on UV and VIS light irradiation. Using a photochromic azobenzene derivative, we have demonstrated that the ATPase activity of the motor protein kinesin is photoregulated.

Fluoroaluminate and fluoroberyllate are potent inhibitors of the ATPase activity of myosin. Inhibition requires the presence of ADP, and much evidence has accumulated to suggest that the tetrahedral fluoroaluminate and fluoroberyllate ions act as phosphate analogues, binding with high affinity at the active site in the position normally occupied by the terminal phosphate of ATP. Both the S1-ADP-fluoroaluminate and the S1-ADP-fluoroberyllate species are thought to resemble kinetic intermediates in the actomyosin ATPase cycle. Characterization of S1-bound fluoroaluminate by 19F NMR is straightforward; a single resonance identified as AlF4- is observed easily [Maruta, S., Henry, G.D., Sykes, B.D., & Ikebe, M (1993) J. Biol. Chem. 268, 7093-7100]. Bound fluoroberyllate, by contrast, was found to give rise to four separate peaks: a downfield pair at -80 and -83.5 ppm and an upfield pair at -101.5 and -103 ppm, suggesting the existence of four distinct types of S1-ADP-fluoroberyllate complex. The relative intensities of the bound resonances can be altered by changing rhe F:Be ratio during complex formation. Integration of a spectrum acquired in the presence of a fluorine-labeled nucleotide derivative, 3'(2')-O-(4-fluorobenzoyl)-ADP, in place of ADP yielded a bound fluoride to nucleotide ratio of 1.7-1.9 to 1, showing that the major bound fluoroberyllate species cannot be BeF3- as is usually thought. It is proposed that the bound fluoroberyllates correspond to the neutral species BeF2(H2O)2 and BeFOH(H2O)2 and the negatively charged species [BeF2OH.H2O]- and [BeF3.H2O]-, although other possibilities are discussed.

Ionic liquids (ILs) are considered to be green solvents because of their non-volatility. Although ILs are relatively safe in the atmospheric environment, they may be toxic in other environments. Our previous research showed that the cytotoxicity of ILs to biological organisms is attributable to interference with cell membranes by IL insertion. However, the effects of ILs on ion channels, which play important roles in cell homeostasis, have not been comprehensively studied to date. In this work, we studied the interactions between ILs and lipid bilayer membranes with gramicidin A ion channels. We used two methods, namely electrical and fluorescence measurements of ions that permeate the membrane. The lifetimes of channels were increased by all the ILs tested in this work via stabilizing the compressed structure of the lipid bilayer and the rate of ion flux through gA channels was decreased by changing the membrane surface charge. The former effect, which increased the rate of ion flux, was dominant at high salt concentrations, whereas the latter, which decreased the rate of ion flux, was dominant at low salt concentrations. The effects of ILs increased with increasing concentration and alkyl chain length. The experimental results were further studied using molecular dynamics simulations.