Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric thick filament associated protein that is critically important to normal cardiac structure and function. The importance of cMyBP-C is emphasized by mutations to cMyBP-C being a leading cause of hypertrophic cardiomyopathy. Despite being a key regulator of cardiac contractility, the molecular mechanism by which cMyBP-C modulates actomyosin force and motion generation is far from certain. Although cMyBP-C's N-terminal domains can bind to actin and the myosin head region, it is not known which of these binding partners is physiologically relevant and whether these binding partner interactions modulate cardiac contractility by directly affecting actomyosin power generation or indirectly by altering Ca2+-dependent thin filament activation. With phosphorylation of cMyBP-C's N terminus occurring in response to -adrenergic stimulation, phosphorylation may offer a measure of cMyBP-C functional tunability in order to enhance cardiac contractility. We propose the following three specific aims. Aim 1 tests the hypothesis that cMyBP-C's thin filament activation and actomyosin power inhibition are independent mechanisms associated with a specific binding partner. Thus, cMyBP-C binding partner interactions will be determined using state-of-the-art molecular biophysical approaches in simplified in vitro model systems (e.g. single molecule FRET) and in situ within myofibrils (super-resolution STORM microscopy). Structural mutagenesis of cMyBP-C to ablate binding partner sites of interaction in both expressed N-terminal fragments and in mutant cMyBP-C from transgenic mice will help link cMyBP-C's functional capacities to its interaction with either the thin filament or the myosin head region. Aim 2 tests the hypothesis that cMyBP-C activates the thin filament directly through a mechanism similar to calcium activation. Thus, we have developed an in vitro single thin filament activation assay to monitor the molecular sequence of events by which expressed fluorescently-labeled N-terminal fragments of cMyBP-C initiate the cooperative recruitment of fluorescently- labeled myosin molecules to the thin filament. Aim 3 tests the hypothesis that phosphorylation of cMyBP-C tunes cMyBP-C's modulation of contractility through alterations in cMyBP-C's molecular mechanics, which in turn alters its binding partner interactions. Cardiac tissue and native thick filaments from transgenic mice expressing mutant cMyBP-C as well as expressed N-terminal fragments with one or more serines replaced by non-phosphorylatable alanines or aspartic acids (phosphomimetics) will be used in assays described in Aims 1 and 2 to characterize the effect of site-specific phosphorylation. Using atomic force microscopy, we will characterize possible mechanisms by which phosphorylation affects M-domain molecular mechanics and structure, thus modulating cMyBP-C function. With the knowledge and understanding of cMyBP-C function derived from these collective studies, targeted therapies directed at cMyBP-C binding partner interactions may be developed to help modulate and to improve cardiac performance in the failing heart.