Dual Regulatory Functions of the Thin Filament Revealed by Replacement of the Troponin I Inhibitory Peptide with a Linker

Striated muscles are relaxed under low Ca2+ concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca2+ concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca2+ dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca2+ rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca2+ concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca2+ sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca2+, and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca2+. The overall results indicate that cooperative myosin binding to Ca2+-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca2+ binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.