We have shown previously that the hypertrophic cardiomyopathy mutant HcTnC D145E lacks α-helical content compared to WT and confers greater Ca2+ sensitivity on regulated thin filaments at 21°C. We hypothesize that the functional alterations are caused by protein instability. Here, using spectroscopy and skinned-fiber assays, we examined the thermodynamic and functional parameters of HcTnC D145E at physiologically relevant temperatures. Based on circular dichroism, isolated D145E lost more of its secondary structure than did WT at high temperatures (30-45°C). Melting temperatures Tm for WT and D145E were 71.8±0.2 vs 51.5±0.8°C in Mg2+ (1 mM free)+EGTA and 78.2±0.2 vs 55.7±1.0°C in 1 mM free Mg2+, pCa 4. In 3M urea (with and without Ca2+/Mg2+), D145E lost nearly all secondary structure at 10°C while WT began to unfold only at 45-50°C. using bis-ANS, a fluorescent probe that binds to hydrophobic surfaces, we found that apoD145E was structurally more open than WT, while Ca2+, which opens hydrophobic pockets in apoTnCWT, had no additional effect on D145E. The Ca2+ affinity increment (ΔpCa50) (=pCa50D145E-pCa50WT) of isolated D145E vs WT double-labeled with 2-(4'-(2''-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid (IAANS) was greater at higher temperatures, i.e. 0.11, 0.25 and 0.66 log units at 21°, 30° and 45°C respectively. In cardiac skinned fibers at higher temperatures, Ca2+ binding affinity (-log Ca50) was further increased for D145E (from pCa50 5.55±0.01 at 15°C to 6.34±0.02 at 30°C) than for WT (pCa50 5.39±0.01 at 15°C to 5.96±0.04 at 30°C), so that ΔpCa50 measured at different temperatures increased in a linear fashion. Maximal tension (P0) was markedly lower for D145E compared to WT at 30°C. These data indicate that D145Es instability dictates its impaired function within the physiological milieu. NIH-HL103840 (JRP)