Time-resolved tryptophan emission study of cardiac troponin I.
We have carried out a time-resolved fluorescence study of the single tryptophanyl residue (Trp-192) of bovine cardiac Tnl (CTnl). With excitation at 300 nm, the intensity decay was resolved into three components by a nonlinear least-squares analysis with lifetimes of 0.60, 2.22, and 4.75 ns. The corresponding fractional amplitudes were 0.27, 0.50, and 0.23, respectively. These decay parameters were not sensitive to complexation of CTnl with cardiac troponin C (CTnC), and magnesium and calcium had no significant effect on the decay parameters. After incubation with 3':5'-cyclic AMP-dependent protein kinase, the intensity decay of CTnl required a fourth exponential term for satisfactory fitting with lifetimes of 0.11, 0.81, 1.95, and 6.63 ns and fractional amplitudes of 0.06, 0.37, 0.27, and 0.29, respectively. When bound to CTnC, the intensity decay of phosphorylated CTnl (p-CTnl) also required four exponential terms for satisfactory fitting, but the longest lifetime increased by a factor of 1.7. The decay parameters obtained from the complex formed between p-CTnl and CTnC were not sensitive to either magnesium or calcium. The anisotropy decay was resolved into two components with rotational correlation times of 0.90 and 23.48 ns. Phosphorylation resulted in a decrease of the long correlation time to 14.61 ns. The anisotropy values recovered at zero time suggest that the side chain of the Trp-192 had considerable subnanosecond motional freedom not resolved in these experiments. Within the CTnl.CTnC complex, the unresolved fast motions appeared sensitive to calcium binding to the calcium-specific site of CTnC. The observed emission heterogeneity is discussed in terms of possible excited-state interactions in conjunction with the predicted secondary structure of CTnl. The loss of molecular asymmetry of cardiac troponin I induced by phosphorylation as demonstrated in this work may be related to the known physiological effect of beta-agonists on cardiac contractility.
Liao
R
R
Graduate Program in Biophysical Sciences, University of Alabama, Birmingham 35294-2041.
Wang
C K
CK
Cheung
H C
HC
D000818:Animals; D002118:Calcium; D002417:Cattle; D004533:Egtazic Acid; D005454:Fluorescence Polarization / Q000379:methods; D006352:Heart Ventricles; D007700:Kinetics; D008274:Magnesium; D009206:Myocardium; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014336:Troponin / Q000737:chemistry* / Q000302:isolation & purification; D019210:Troponin I; D014364:Tryptophan
D014336:Troponin; D019210:Troponin I; D004533:Egtazic Acid; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium