Molecular Motor Group

■Object

Myosin is an enzyme which moves actin filaments utilizing energy released by hydrolysis of ATP. Myosin is often referred to as a molecular motor, because it converts chemical energy to mechanical energy. In this respect, myosin is functionally similar to artificial motors such as automobile engines. Myosin has several remarkable features that are not seen in artificial motors. For instance, myosin is smaller than automobile engines by a factor of 1022, functions as a single molecule, converts energy very efficiently, and works at room temperature without becoming hot. Thus it is evident that myosin uses a totally novel molecular principle for the chemical to mechanical energy conversion. However, we still do not know how myosin works. Understanding its molecular mechanism is not only extremely interesting and challenging as basic science, but should be useful for future designing of novel artificial micromachines.

■Results in Fiscal Year

(1) Swinging lever arm model

A currently popular model hypothesizes that myosin motors changes the shape when strongly bound to actin filaments, and this conformational change generates force and relative sliding between actin filaments and the myosin backbone (Figure 1, left). This model is called the ﾒswinging lever arm modelﾓ. To test this model, we are combining recombinant myosin expression technology and in vitro movement assay, which allows quantitative assay of motility of recombinant mutant myosins under microscopes. Here is how we are trying to test the swinging lever arm model using these tools.

A man walks by swinging motion of legs, similarly to how myosin moves according to the swinging lever arm model. The step size of walking men should be proportional to the length of legs if the swinging angle of legs are the same. Therefore, if the step frequencies are unchanged, walking speeds should be proportional to the leg lengths. By analogy, myosinﾕs speed should be proportional to the length of light chain binding region or the ﾒneckﾓ region, which corresponds to legs, if the swinging lever arm model is correct. Conversely, if one can demonstrate a linear relationship between the speed and the length of neck region, that would be a strong support for the model. We have therefore created three mutant myosins with altered length of the neck region and measured their sliding velocities under microscopes. We were able to demonstrate a good linear relationship between the two parameters (Figure 2). This result strongly supports the swinging lever arm model.

We have launched a new project to understand how hydrolysis of ATP is coupled to this intramolecular conformational change that leads to the swinging motion of the neck region.

(2) The linear motor model

A radically different model assumes that myosin motor slides along actin filaments (Figure 1, right). This model is called the ﾒlinear motor modelﾓ. Small particles such as protein molecules are fiercely agitated by thermal movement of surrounding water molecules. Thus one cannot view action of molecular motors analogously to macroscopic artificial machines. It should be more natural to assume that actin and myosin change their relative position through repetition of binding and detaching driven by thermal agitation. At first glance, one might think that the probability of moving both directions are the same and therefore there should be no net displacement. To solve this problem, we hypothesize that ATP drives a conformational change within the myosin motor domain , which allows transfer of the energy to actin. This energy is assumed to create asymmetry of actinﾕs surface state (hydrophobicity) with respect to myosin, leading to unidirectional drift of myosin along the actin surface. To test this possibility, we have been analyzing mobility of water molecules in solutions containing myosin motors by using microwave dielectric measurements, which yields information about the surface hydrophobicity of the solute. We were able to demonstrate changes of surface hydrophobicity of the myosin motor during the course of ATP hydrolysis and importantly, the magnitude of the changes was consistent with the hypothetical conformational changes within the myosin motor (Figure 3). This results not only explains why the strength of interaction between actin and myosin decreases in the presence of ATP, but also indicates that hydrophobicity change of myosin has a potential to generate sufficient force to drive the linear movement of actin.

Figure 1. Two popular models for production by myosin.

Figure 2. A linear relationship between the neck length of mutant myosins and their sliding velocities.

Figure 3.