Thin filament-linked actin-binding proteins, troponin and tropomyosin, control actomyosin-based muscle contraction in cardiac and skeletal muscles. To elucidate mechanisms of muscle thin filament function at a fundamental molecular level, it is crucial to determine the changing structural interactions of these regulatory proteins that control muscle cooperative activation and relaxation via allosteric communication pathways between filament components. It follows that disease-related myofibrillar protein mutants can perturb muscle on-off switching by causing an imbalance in troponin-tropomyosin interactions on actin which, in turn, destabilizes relaxed or active states and the transitions between them. It is our premise that early stage intervention to correct such imbalances is paramount in diminishing or reversing resulting inexorable disease progression. In the current work, we will address these imbalances by taking a multifaceted structural approach to elucidate the mechanism of thin filament regulation and thus establish root causes of these perturbations. To accomplish this goal: 1. We will use cryo-electron microscopy, coupled with 3D-image reconstruction, to establish regulatory transitions of troponin and tropomyosin as well as test the impact of myosin-binding on thin filament actin and tropomyosin. 2. We will refine this experimental approach with computational tools that we have pioneered to bring cryo-EM structures even closer to an atomic level using protein-protein docking protocols and molecular dynamics. 3. We will compare structural interactions that occur in normal thin filaments with those in filaments containing mutant proteins linked to myopathies in order to assess how mutation-linked aberrant physiology can link to myopathology, while localizing druggable target pockets at protein-protein interfaces. To achieve our aims, (1) we will focus on identifying structural domains at the interface between of troponin subunit-T and actin-tropomyosin (Specific Aim 1); (2) we will reveal the structural mechanism used by regulatory domains of troponin subunit-I to trap tropomyosin in its relaxed-state position on actin (Specific Aim 2); (3) we will determine the impact of myosin structural interactions on actin-tropomyosin, less recognized but significant effectors of thin filament regulation (Specific Aim 3). The influence of myopathic-linked mutations in troponin, tropomyosin, actin and myosin will not only be predicted and tested structurally but assayed functionally by measuring in vitro motility and contractility in engineered heart tissue. Aiming to develop tools to counteract regulatory imbalances, we collaborate with associates at the Boston University Central for Molecular Discovery to identify small molecules to be trapped at druggable interfaces along thin filaments in order to potentially manipulate cooperative, regulatory pathways. Thus, our work on the molecular regulation of cardiac and skeletal muscle thin filaments an...