Distance measurements in cardiac troponin C.
Intramolecular distance measurements were made in cardiac troponin C (cTnC) by fluorescence energy transfer using Eu3+ or Tb3+ as energy donors and Nd3+ or an organic chromophore as acceptors. The laser-induced luminescence of bound Eu3+ is quenched in Eu1Nd1cTnC with a lifetime of 0.328 ms, compared with 0.43 ms for Eu2cTnC. The enhanced decay corresponds to an energy transfer efficiency of 0.25, or a distance of 1.1 nm between the two high affinity sites. We have also labeled cTnC with 4-dimethylaminophenylazophenyl-4'-maleimide (DAB-Mal) at the two cysteine residues (Cys-35 and Cys-84). Energy transfer measurements were carried out between Tb3+ bound to the high affinity sites and the labels attached to the domain containing the low affinity site. Upon uv irradiation at pH 6.7, Tb1cTnCDAB emits tyrosine-sensitized Tb3+ luminescence that decays bioexponentially with lifetimes of 1.29 and 0.76 ms. The shorter lifetime is ascribed to energy transfer from Tb3+ to the DAB labels, yielding an average distance of 3.4 nm between the donor and the acceptors. At pH 5.0, however, the luminescence decays exclusively with a single lifetime of 1.31 ms, suggesting that under these conditions all Tb3+ ions are more than 5.2 nm away from the label. Thus cTnC, like skeletal TnC, undergoes a pH-dependent conformational transition which converts an elongated structure at lower pH's to a rather compact conformation in a more physiological medium.
Wang
C L
CL
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Leavis
P C
PC
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003545:Cysteine; D004735:Energy Transfer; D005063:Europium; D007700:Kinetics; D008163:Luminescent Measurements; D009206:Myocardium / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013725:Terbium; D014336:Troponin / Q000302:isolation & purification / Q000378:metabolism*; D019209:Troponin C; D004124:p-Dimethylaminoazobenzene / Q000378:metabolism / Q000031:analogs & derivatives
D000345:Affinity Labels; D014336:Troponin; D019209:Troponin C; D013725:Terbium; D005063:Europium; C049748:4-dimethylaminophenylazophenyl-4'-maleimide; D004124:p-Dimethylaminoazobenzene; D003545:Cysteine