PROPERTIES OF TROPONIN-C ACETYLATED AT LYSINE RESIDUES

We have studied the properties of rabbit skeletal troponin C (TnC) ful ly acetylated at its lysine residues (AcTnC). Acetylation causes a dec rease in thermal stability of both domains of TnC in the absence of Ca 2+. At 25 degrees C, the acetylated C-terminal domain of TnC is almost completely unfolded and the melting temperature of the N-terminal dom ain monitored by far-UV circular dichroism is decreased by 16.3 degree s C. In the presence of 1 mM CaCl2, no cooperative unfolding can be de tected up to 90 degrees C for either TnC or AcTnC. At 25 degrees C, CD spectra show that AcTnC has a slightly lower alpha-helix content in t he absence of Ca2+, but higher in the presence of Ca2+ as compared to unmodified TnC. Acetylation causes a 3.5-fold increase in affinity for Ca2+ at the low-affinity sites and a 2-fold decrease at the high-affi nity sites. Polyacrylamide gel electrophoresis under nondissociating c onditions (no SDS, no urea, pH 8.6) indicates that acetylation has lit tle effect on the apparent affinity of TnC for troponin I; however, th e binding of the acetylated peptides corresponding to the N-terminal d omain of TnC to troponin I is significantly stronger than that of the unmodified peptides. Troponin T binding to AcTnC is significantly enha nced, the altered properties of the N-terminal domain being predominan tly responsible for the increase. Titration of the ATPase activity of TnC-depleted myofibrils with AcTnC or native TnC indicates that acetyl ation increases TnC's affinity for myofibrils in the presence of Ca2similar to 6 times; at saturation the ATPase activity is the same for the two forms of TnC. The Ca2+ dependence of the ATPase activity of my ofibrils containing AcTnC is shifted to lower Ca2+ concentrations, con sistent with the higher Ca2+ affinity of AcTnC at the low-affinity sit es. These data indicate that positively charged lysine side chains, es pecially those located in the N-terminal domain, modulate TnC's struct ural stability and interactions with Ca2+ and other troponin component s.