9505458 Vibert The goals of this project are to determine the structures of muscle thick and thin filaments and to interpret them in terms of the recently determined atomic structures of the cmmponent proteins. The head region of the myosin molecule, which contains its enzymatic site and which interacts with actin to produce force, can now be produced in native or modified form by molecular biological techniques, and the atomic structure of a myosin head has been determined by x-ray crystallography. The structure of native and mutant myosin heads attached to actin filaments will be determined by electron microscopy of frozen-hydrated samples combined with three-dimensional image reconstruction. Structure determination at 10-15A resolution permits elements of protein secondary structure such as -helices can be recognized, but even at lower resolution, fitting known atomic structures into the envelope of the reconstructions allows them to be interpreted in detail. Of particular interest is the conformation of the regulatory domain of the myosin head and how this changes when regulatory switching occurs. These structural methods will also be employed to study regulation of contraction by the tropomyosin-troponin complex bound to actin in the thin filaments; reconstructions will be interpreted in terms of atomic models of actin and tropomyosin. Of particular interest is establishing the location and possible movements of troponin in thin filaments at different stages of regulatory switching. Electron cryo-microscopy and image reconstruction will also be used to determine how myosin subfilaments are incorporated into native thick filaments. Among the proteins known to associate with myosin are those of the titin family; vertebrate titin plays an elastic role in the muscle cell, and may also define how thick filaments align during muscle development. Co-assembly of myosin and smaller titin isoforms believed to have structural and regulatory roles will be exami ned by electron microscopy to establish which features of native filament structure depend on the interactions between these proteins. %%% Muscle contraction requires the organization of contractile and regulatory proteins into large complex assemblies. To understand contraction in molecular terms, it is therefore necessary to know not only the structures of the individual proteins, but also how the proteins assemble into the larger structural units found in the cell. Two aspects of muscle cell organization and of the control of contraction are emphasized: the mechanisms by which protein subunits regulate myosin-actin interaction and thus contraction, and the process by which contractile and accessory proteins assemble to form the filaments of the muscle cell. The size and complexity of the protein filaments in muscle means that the method of choice for analyzing their structures is electron microscopy. In cases where the filament components are symmetrically arranged, image analysis offers a further powerful mathematical and computational approach to determining their three-dimensional structure and arrangement. When atomic-level information about the separated components is available, it can be incorporated to produce a highly detailed picture of the functional units in the muscle cell, and thus open a path to a new understanding of muscle contraction at the molecular level. ***
