This proposal is concerned with new experimental approaches to understanding the basic molecular mechanism of cellular motility. It is built on the established work of six laboratories, each bringing a separate area of expertise to this problem. Two types of motors will be studied: the interaction of the kinesin family motors with microtubules; and the interaction of myosin with actin. Some structural and biochemical similarities suggest that the molecular mechanisms of these two motile systems may share common features. Each system has its particular strengths and limitations, and techniques developed for the study of one system, can often be applied to the other. Central to the study of microtubule motors will be the determination of the three-dimensional structure of one of them, the ncd protein, using X-ray crystallography. However information on the structural changes that occur during the generation of motion is also necessary to understand the mechanism of this motor. To obtain this information we will use neutron diffraction and spectroscopic probes to measure the changes occurring in the structure of microtubule motors upon interaction with nucleotides and microtubules. The energetics associated with these interaction will also be determined. The force generated by single microtubule motors will be measured, bringing new insight to their motor mechanics. Using mutagenesis, we will construct novel monomeric and dimeric molecules to determine whether the two-headed nature of these molecules is crucial to their ability to generate motion. These studies will be facilitated by a collaboration between laboratories that have developed many of these techniques for the study of myosin, and those now studying the microtubule motors. Our studies of the actomyosin system are motivated by the recently published structures of both actin and myosin, which have led to a hypothesis for how force is generated at the molecular level. To provide critical data to test this hypothesis, we will use a number of genetic and physical chemical techniques. Spectroscopic probes will monitor the conformation and orientation of key structural elements in the myosin molecule. These elements include two deep clefts, which are involved in the binding of actin and nucleotides, and the neck region, which may act as a lever system that amplifies conformational changes generated by nucleotide and actin binding. We will measure shape changes in the myosin head using neutron and X-ray diffraction. Careful measurements of actin-myosin affinity under a variety of conditions will help establish which charged residues are involved in this interaction. The above approaches will be applied to mutants of myosin which are arrested at different points in the cycle. These mutants will provide the ability to establish the structure of intermediates of the cycle, many of which are normally present only transiently. In summary, our goal of understanding the mechanism of biological motors requires: a) a determination of their atomic resolution structures, b) knowledge of how these structures change in specific states and c) measurements of the mechanics and energetics associated with these states. In this program we have brought together a unique group of investigators that can extend our knowledge in each of these areas leading to a better picture of the molecular mechanism of these two motors that produce force and motion in all eukaryotic cells.
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