Project Summary: Hypertrophic cardiomyopathy (HCM) is a complex genetic cardiac disorder that affects ~1/300 – 1/500 individuals worldwide. A common clinical manifestation of patients with HCM is an impairment in left ventricular relaxation (diastolic dysfunction). Beta-adrenergic stimulation is a key regulator of diastolic performance. During beta- adrenergic stimulation protein kinase A (PKA) mediates phosphorylation of a variety of sarcomeric targets, including cardiac troponin I (cTnI) at serine 23/24 (Ser23/24). This phosphorylation event results in a significant increase in relaxation (or de-activation) rates at the myofilament level. Previous work has shown that this observation is due to increases in calcium dissociation rate from the cardiac thin filament. Additionally, some thin filament HCM mutations have been shown to exhibit an impaired response to phosphorylation of Ser23/24 in ATPase assays and force-pCa measurements. While extensive work by several groups has investigated the structural basis for this increase in calcium dissociation rate, all previous studies, to the best of our knowledge, lack the key thin filament binding partners actin and tropomyosin, crucial components for allosteric regulation of relaxation. In this proposal we will perform TR-FRET experiments to assess both intramolecular and intermolecular interactions between the N-terminus of cTnI and C-terminus of cTnI and the N-terminus of cTnI and Site II of cTnC in the presence and absence of phosphorylation at Ser23/24. The experimental design will provide distances that we will employ in our atomistic thin filament model. We will then use stopped flow fluorescence anisotropy in order to probe transitions in dynamic behavior in the C-terminus of cTnI when Ser23/24 is phosphorylated as calcium dissociates from the cardiac thin filament. We hypothesize that phosphorylation of Ser23/24 will alter the rate at which these transitions occur and that these mechanisms may be altered by HCM causative mutations. To explore the possibility that the degree of observed diastolic impairment (and potentially the severity of the end HCM phenotype) may be mutation-specific, we propose to investigate the molecular effects of 3 independent known cTnI mutations at residue R145 in cTnI. This mutational hotspot includes HCM- linked mutations R145G, R145Q and the restrictive cardiomyopathy (RCM) mutation R145W. We will couple structural data from TR-FRET experiments to changes in calcium dissociation rate to investigate how structural changes impact function and if the diastolic dysfunction is additive in the presence of Ser23/24 phosphorylation. We will employ metadynamics simulations to obtain free energy changes and identify specific changes in interactions that occur from these mutations and phosphorylation as well as in the two calcium states (on and off). The in-vitro—in-silico coupled approaches proposed in this application will provide atomic level resolution of the structural ch...