There is now strong if not overwhelming evidence that myosin-based movement results largely from a change in lever arm angle while the motor domain is attached to actin, in what is called the tilting lever mechanism. What is now required is to understand this mechanism in considerably greater detail, especially the correlation between different biochemical and structural states, details of the movement mechanisms in different myosin types, and whether the tilting mechanism alone provides a complete explanation of movement. A major aspect of our work will be to use time-resolved electron cryo-microscopy, which allows specimens to be examined under conditions very close to native and with excellent time resolution. We will use new image processing methods and automated electron microscopy to obtain the very large amounts of data that are necessary for high resolution images. Our goal will be to achieve <1 nm resolution, which allows internal alpha-helices in molecules to be revealed. If this can be done, there is the exciting prospect of seeing the individual elements, several of which are alpha helices, and how they change in different biochemical states to produce movement. Most of our work will use myosin V. Its 'weakly' bound states with actin have a much higher affinity than those of myosin II, allowing study of the pre-power stroke conformation. Moreover, the large size and asymmetry (6 calmodulins in the regulatory domain) make myosin V especially suitable for electron microscopy. However, we will also explore the use of non-muscle forms of myosin II (NMIIA and NMIIB) that have very slow rates of product release in the presence of actin. Our work is directly relevant to a number of diseases involving myosins. Myosin V has been identified as being the gene responsible for the """"""""dilute mutation"""""""" in mice, which has defective transport of pigment to hair follicles, and is also responsible for developmental and neurological disorders. Non muscle myosin II plays key roles in platelet activation and the regulation of blood clotting.
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