Thin filament-linked actin-binding proteins control both actomyosln-based muscle contraction and cytoskeletal formation. In order to understand the normal control of muscle contraction, it is crucial to characterize the structural interactions of thin filament regulatory proteins that influence actin-mvosin association. Using structural analysis, we will examine the architecture of cardiac and skeletal muscle thin filaments at a fundamental level and determine the changing interactions of thin filament-linked proteins that regulate muscle activity. Our principal objective therefore is to solve the atomic structure of the entire thin filament in relaxed and in active muscle. We use state-of-the-art electron microscopv and electron tomographv. coupled with image analysis, 3D reconstruction (3DEM) and computational ehemistrv to establish the macromolecular structure of actin-binding proteins on thin filament actin and thus demarcate molecular contacts of binding proteins with actin at near atomic resolution. Using these techniques: (1) We aim to detennine the structural basis of troponin-tropomyosin regulation of cardiac and skeletal muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments, which are governed by Ca^^-binding to troponin and myosin-crossbridge binding on actin. To accomplish this goal, (a) we will describe the complete atomic structure of tropomyosin and troponintropomyosin on thin filaments by generating single particle and electron tomographic reconstructions at increasingly high resolution and by fitting atomic resolution crystal structures of actin and regulatory proteins into the reconstruction volumes; (b) we will further refine these atomic structures using computational tools to maximize chemical interactions between regulatory proteins and actin; (c) we will use Molecular Dynamics protocols to assess regulatory protein dynamics and likely transitions between regulatory states. (2) Using this same approach, we will test the hypothesis that mutant cardiac troponin and tropomyosin perturb muscle regulation by causing imbalanced protein interactions that alter the regulatory state of thin filaments, and we will determine the underlying structural reasons for such perturbations. (3) We will test the hypothesis that the short molecular overlap domain between adjacent tropomyosins, needed for end-to-end tropomyosin linkage, is vital for tropomyosin strand formation on filaments, and again assess the impact of mutants on this structure. (4) We will complete efforts to test the hypothesis that Myosin-Binding Protein C forms regular and periodic links to thin filaments, which are likely to modulate striated contractile activity.

Studies on tropomyosin- and troponin-regulated filaments, with particular attention devoted to normal and mutant proteins, will lead to an elucidation ofthe molecular regulatory mechanisms governing cardiac muscle contraction. This work is essential for unraveling cardiomvopathic disease processes and revealing key controls for vascular tone and pulmonary ainvay resistance, determinants in, for example, hypertension and asthma. Actin filaments and their associated proteins are major participants in diverse cellular systems having varied function, thus underscoring the broad significance of the proposed work.