The effects of two Leu‐to‐Pro substitutions, L57P and L43P , on structural and functional properties of cardiac tropomyosin

Tropomyosin (Tpm) is an actin‐binding protein that, together with troponin (Tn), mediates Ca 2+ ‐regulation of cardiac muscle contraction. Tpm coiled‐coil dimers bind each other through overlap junctions between their N‐ and C‐termini, forming a continuous strand along the actin filament. Among the many TPM1 mutations identified in cardiac Tpm (Tpm1.1), few substitute canonical residues with proline, the amino acid most disruptive to coiled‐coil structure. We examined properties of recombinant cardiac Tpm with L43P or L57P substitutions in both chains and compared them with wild‐type (WT) Tpm using differential scanning calorimetry, viscometry, molecular dynamics (MD) simulations, and an in vitro motility assay. Both mutations markedly destabilized the N‐terminal part of the Tpm molecule. In MD simulations, Pro43 and Pro57 disrupted nearby backbone hydrogen bonds, and Pro43 promoted N‐terminal unfolding, demonstrating a long‐range effect within supercoiled Tpm molecules. Both substitutions strongly reduce Tpm's affinity for F‐actin in the absence of Tn. In addition, viscometry showed that the L43P Tpm variant polymerizes less efficiently than WT and L57P Tpm, resulting in a reduced ability to assemble into a continuous strand along an actin filament. The L57P substitution increases maximum sliding velocity of thin filaments in in vitro motility assay and enhances Ca 2+ sensitivity of actin–myosin interaction, a feature commonly associated with hypertrophic cardiomyopathy. In contrast, the L43P substitution hinders the formation of fully regulatory‐competent thin filaments and severely impairs the Ca 2+ ‐regulatory function of reconstructed thin filaments in vitro . Our study reveals distinct mechanisms of pathogenic effects for these two largely similar amino acid substitutions.