title: Properties of troponin C acetylated at lysine residues.
abstract: We have studied the properties of rabbit skeletal troponin C (TnC) fully acetylated at its lysine residues (AcTnC). Acetylation causes a decrease in thermal stability of both domains of TnC in the absence of Ca2+. At 25 degrees C, the acetylated C-terminal domain of TnC is almost completely unfolded and the melting temperature of the N-terminal domain monitored by far-UV circular dichroism is decreased by 16.3 degrees C. In the presence of 1 mM CaCl2, no cooperative unfolding can be detected 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 the 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-affinity sites. Polyacrylamide gel electrophoresis under nondissociating conditions (no SDS, no urea, pH 8.6) indicates that acetylation has little effect on the apparent affinity of TnC for troponin I; however, the binding of the acetylated peptides corresponding to the N-terminal domain of TnC to troponin I is significantly stronger than that of the unmodified peptides. Troponin T binding to AcTnC is significantly enhanced, the altered properties of the N-terminal domain being predominantly responsible for the increase. Titration of the ATPase activity of TnC-depleted myofibrils with AcTnC or native TnC indicates that acetylation increases TnC's affinity for myofibrils in the presence of Ca2+ approximately 6 times; at saturation the ATPase activity is the same for the two forms of TnC. The Ca2+ dependence of the ATPase activity of myofibrils containing AcTnC is shifted to lower Ca2+ concentrations, consistent with the higher Ca2+ affinity of AcTnC at the low-affinity sites. These data indicate that positively charged lysine side chains, especially those located in the N-terminal domain, modulate TnC's structural stability and interactions with Ca2+ and other troponin components.
authors.affiliation: Boston Biomedical Research Institute, Neurology Service, Massachusetts General Hospital, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Grabarek
authors.forename: Y
authors.initials: Y
authors.lastname: Mabuchi
authors.forename: J
authors.initials: J
authors.lastname: Gergely
mesh_terms: D000107:Acetylation; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D004355:Drug Stability; D004563:Electrochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D018482:Muscle, Skeletal / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011817:Rabbits; D013816:Thermodynamics; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T
chemical_list: D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D000251:Adenosine Triphosphatases; D008239:Lysine; D002118:Calcium