A fluorescent probe study of Ca2+ binding to the Ca2+-specific sites of cardiac troponin and troponin C.
Cardiac troponin C (C-TnC) was labeled with the sulfhydryl-specific fluorescent probe molecule 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid at cysteine 35 and 84 to produce C-TnCIA. This modified protein binds Ca2+, undergoes Ca2+-induced increases in alpha helix, and forms a complex with other troponin subunits as does unlabeled C-TnC. C-TnCIA undergoes a small fluorescence decrease with Ca2+ or Mg2+ binding to the two high affinity Ca2+-Mg2+ sites of C-TnC and a large biphasic approximately 2.1-fold fluorescence increase with Ca2+ binding to two lower affinity Ca2+-specific sites with KCa of approximately 4.5 X 10(5) M-1 and approximately 5 X 10(2) M-1. C-TnCIA was formed in a complex with troponin I (TnI) and troponin T to form C-TnIA. This fluorescent reconstituted whole troponin undergoes a 25% decrease with Ca2+ binding to a Ca2+-specific site of KCa approximately 3 X 10(6) M-1. C-TnC, therefore, contains a single Ca2+-specific site of approximate equal affinity as the two Ca2+-specific regulatory sites of skeletal TnC. This Ca2+-specific site in C-TnC (like its two corresponding sites in S-TnC) undergoes an approximate 10-fold increase in affinity in whole troponin or when TnC is complexed with TnI. Since the two Ca2+-specific sites in skeletal troponin have been shown to be the regulatory sites of skeletal muscle contraction we suggest that this single Ca2+-specific site, of equal affinity, in C-TnC is the regulatory site of cardiac muscle contraction.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D009124:Muscle Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D005456:Fluorescent Dyes; D009124:Muscle Proteins; D009282:Naphthalenesulfonates; D014336:Troponin; D019209:Troponin C; C011488:IAANS; D002118:Calcium