Differential recovery of Ca2+ binding activity in mutated EF-hands of cardiac troponin C.
Previous studies showed that conversion of the first Ca2+ ligand in Ca(2+)-binding sites III and IV from Asp to Ala decreased the affinity of cardiac TnC (cTnC) for the thin filament. Here, the functional consequences of mutation of the second ligand in the Ca(2+)-binding sites of cTnC were determined. Equilibrium dialysis and Tyr fluorescence studies showed that conversion of the second Ca2+ ligand to Ala (Asp-67, site II; Asn-107, site III; and Asn-143, site IV) inactivated all three Ca(2+)-binding sites in the free protein. Ca2+ binding to the mutated site II was not recovered upon association with a troponin complex, and proteins with this mutation were unable to regulate Ca(2+)-dependent ATPase activity in TnC-extracted myofibrils. However, Ca2+ binding was recovered at the mutated sites III and IV under the same conditions. Sequential addition of active and inactive cTnC proteins in a myofibril ATPase assay suggested that that Mg2+ binding was not recovered and that the recovered Ca2+ affinity of the mutated sites III and IV was much lower than that of the wild type in that the Ca2+ concentrations required for apparent thin filament binding by proteins containing mutations at sites III and/or IV were significantly greater than that required for the wild-type protein.
Dotson
D G
DG
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Putkey
J A
JA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D005453:Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D011817:Rabbits; D014336:Troponin / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C; D014443:Tyrosine / Q000737:chemistry
D014336:Troponin; D019209:Troponin C; D014443:Tyrosine; D000251:Adenosine Triphosphatases; D002118:Calcium