The cardiac troponin I (cTnI) mutation R145G, associated with hypertrophic cardiomyopathy, increases the calcium sensitivity of contraction and alters the kinetics of relaxation. It is unclear whether this results from altered calcium binding of cTn, altered interaction with troponin C (cTnC), or both. It is also unclear how this alters protein kinase A (PKA) mediation of cTn function and myofibril relaxation. We are studying this using an experimental and computational approach. Steady-state fluorescence experiments indicate a significant increase in calcium binding affinity of cTn with R145G vs. wild-type (WT) cTnI. PKA phosphorylation of cTnI resulted in a similar reduction in this affinity for both cTn complexes. Interaction of cTnC with cTnI (C-I) was greater with R145G vs. WT-cTnI. PKA treatment of cTnI weakened C-I interactions for WT, but not R145G-cTnI. PKA treatment of WT-cTnI exchanged rat ventricular myofibrils significantly affected the slow phase of relaxation, increasing the slow phase rate (kREL,slow) and decreasing slow phase duration (tREL,slow). These effects were larger at submaximal calcium levels. Interestingly, R145G-cTnI blunted PKA mediated effects. Molecular dynamics simulations of the crystal structure of cTn were used to investigate the molecular contacts between the cTnI switch peptide region and cNTnC for WT and R145G-cTnI. Preliminary results suggest a profound effect of the mutation on the interaction of residues 147-149 cTnI with cNTnC. For R145G cTn, switch peptide residues 148 and 149 lose most of their strong contacts with cNTnC, and residue 147 forms strong contacts not observed with WT-cTnI. The results suggest the C-I interaction as a molecular level site of R145G effects on relaxation of cardiac muscle. HL65497 (MR), AHA 7400069 (VR), GM103426-19S1 (PA), AHA 12POST11570005 (SL), GM31749 (JAM), NSF MCB 1020765 (JAM), HHMI (JAM), SDSC (JAM), CTBP (JAM).