Regulation of myocardial contractility is influenced by numerous signaling agents. One of the key methods for modification of cardiomyocyte function is phosphorylation of cardiac troponin (cTn), the Ca2+-dependent regulatory switch that controls thin filament activation, by isoforms of protein kinase C (PKC). PKC has been shown to phosphorylate the tropomyosin binding subunit of cTn (cTnT) at Thr-195, Thr-199, Thr-204 and Thr-285 (rat cTnT). The effect of these phosphorylations on cTn structure and dynamics remains largely unknown. To ascertain the site specific effects of PKC phosphorylation of cTnT on thin filament regulation, pseudo-phosphorylation of cTnT was combined with Forster resonance energy transfer (FRET) to monitor the transition between the Ca2+ and Mg2+ states of cTn. Single-cysteine cTnI mutants were labeled with IAEDENS at either Cys-151 or Cys-167, and a single-cysteine cTnC mutant was labeled at Cys-89 with DDPM. Different combinations of mutant cTnT and fluorophore labeled cTnI and cTnC were then incorporated into reconstituted thin filaments, which were subjected to calcium titrations, time resolved FRET structural measurements, and stopped flow kinetic measurements. Titrations showed a consistent pseudo-phosphorylation dependent increase in pCa50 values, and kinetics experiments showed decreased rates of Ca2+ dissociation in pseudo-phosphorylated mutants. Additionally, Mg2+ distances between Cys-167 of cTnI and Cys-89 of cTnC were reduced in the presence of pseudo-phosphorylation. Thus our results suggested that pseudo-phosphorylation of cTnT leads to sensitization of the Ca2+-dependent conformational transitions involved in cTn regulatory switching. However, a prior in situ study demonstrated a pseudo-phosphorylation dependent decrease in EC50 (JBC vol. 278, pp. 35135-44). These results may suggest PKC mediated phosphorylation of cTnT may represent a post-translational modification where cTn regulatory switching is sensitized to Ca2+, but also partially uncoupled from Tm movement.