The structural basis for the conversion of chemical energy to mechanical movements is a major unresolved problem of muscle contraction and myosin based motility. Using the newly developed structural method of cryo atomic force microscopy, which can provide nm resolution with macromolecular complexes without metal coating, we focus on three major aspects of smooth muscle contraction in this project. The first objective is to search for the predicted structural changes and rearrangements in both isolated single myosin molecules and actomyosin complexes, under the conditions corresponding to each major biochemical (ATP hydrolysis) event during the crossbridge cycle.
We aim at obtaining structural information at 2 nm or higher without using averaging methods, and elucidating the crossbridge cycle at the molecular level. The second objective is the interaction of smooth muscle phosphatase with myosin, where the binding stoichiometry will be examine. These studies are expected to provide a structural basis for understanding the function of this important enzyme in smooth muscle regulation. The third objective is the structure of smooth muscle thin filament which plays a pivotal role in the regulation of contraction, yet, very little information is available about its structure, especially when other regulatory components, such as caldesmon and calponin, are present. The problems to be studied here have been central for our understanding of muscle contraction, and have not been resolved, partly because of the limitations of available structural methods. This is the first attempt to apply the cryo atomic force microscopic method to a well defined biological problem, and many of the current technical limitations can be circumvented, providing a unique opportunity to explore the basic properties of mice. In combination with well established smooth muscle physiology, this new approach is well poised to make fundamental contributions to muscle contraction.
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