Properties of Troponin C Acetylated at Lysine Residues
| Autor: | John Gergely, Yasuko Mabuchi, Zenon Grabarek |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Protein Folding
Circular dichroism Lysine Troponin T binding In Vitro Techniques Biochemistry Protein Structure Secondary Troponin C Drug Stability Troponin T Troponin I Electrochemistry Animals Muscle Skeletal Polyacrylamide gel electrophoresis Adenosine Triphosphatases Binding Sites Chemistry Circular Dichroism Acetylation musculoskeletal system Peptide Fragments Troponin Kinetics Biophysics Thermodynamics Calcium Rabbits |
| Zdroj: | Biochemistry. 34:11872-11881 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00037a027 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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