Effect of phosphorylation of cardiac troponin I on the fluorescence properties of its single tryptophan as determined by picosecond spectroscopy

Ronglih Liao; Chien-Kao Wang; Herbert C. Cheung

doi:10.1117/12.17716

1 May 1990 Effect of phosphorylation of cardiac troponin I on the fluorescence properties of its single tryptophan as determined by picosecond spectroscopy

Ronglih Liao, Chien-Kao Wang, Herbert C. Cheung

Author Affiliations +

Proceedings Volume 1204, Time-Resolved Laser Spectroscopy in Biochemistry II; (1990) https://doi.org/10.1117/12.17716
Event: OE/LASE '90, 1990, Los Angeles, CA, United States

Abstract

Cardiac troponin I (CTnI) can be phosphorylated by a c-AMP dependent protein kinase. We have investigated the effect of the phosphorylation on the emission decay properties of its single tryptophan by using a cavity dumped and synchronously pumped dye laser system. At 20°C, τ₁~0.60 ns, τ₂~2.22 ns, and τ₃~4.72 ns. The corresponding fluorescence contributions were 7%, 47%, and 46%, respectively. Upon phosphorylation four lifetimes were observed: τ₁~0.11 ns, τ₂~0.81 ns, τ₃~1.95 ns, and τ₄~6.63 ns, and fractional contributions of the four components were 2%, 16%, 52%, and 30%, respectively. This finding indicates that the environment of the tryptophan is modified by phosphorylation. In the absence of divalent metal ions, the observed three decay times of the CTnI complexed with cardiac troponin C (CTnC) remained unchanged, and addition of Ca²⁺ or Mg²⁺ resulted in only small changes in the lifetimes. When phosphorylated CTnI was complexed with CTnC, a large increase of the longest-lived component was observed: τ₄ > 11 ns with its contribution shifted to 47%. Two rotational correlation times were observed for CTnI: φ₁~0.9 ns and φ₂~ 23.5 ns. These valves increased to t₀~l.2 ns and ~30.1 ns, respectively, for the complex CtnI CTnC. Upon phosphorylation the two correlation times were significantly reduced regardless of whether CTnI was uncomplexed or complexed with CTnC. These results suggest that phosphorylation of CTnI resulted in a significantly more compact structure and enhanced motion of the tryptophan side chain. These structural changes may play a role in the transmission of Ca²⁺ signal in cardiac muscle. Thus, the effect of phosphorylation of CTnI becomes more pronounced when the protein is complexed with CTnC. These results suggest that there was likely a fluorophore heterogeneity which may arise from differences in conformation, environment, and/or different deactivation pathways for the excited state of the fluorophore.

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Ronglih Liao, Chien-Kao Wang, and Herbert C. Cheung "Effect of phosphorylation of cardiac troponin I on the fluorescence properties of its single tryptophan as determined by picosecond spectroscopy", Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); https://doi.org/10.1117/12.17716

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