Troponin-C-mediated calcium-sensitive changes in the conformation of troponin I detected by pyrene excimer fluorescence.
Troponin I (TnI) from rabbit white skeletal muscle was labeled at cysteines 48 and 64 with the fluorescent reagent N-(1-pyrene)maleimide. The fluorescence spectra of pyrene-labeled TnI (pyr-TnI) exhibit peaks characteristic of pyrene in its monomeric form and an additional peak resulting from formation of excited dimers (excimers), indicating that the labeled cysteines are close together. Formation of a pyr-TnI-TnC complex in the absence of Ca2+ has little effect on the spectrum, but when Ca2+ is bound to the low-affinity sites of TnC there is a substantial decrease in excimer and a corresponding increase in monomer fluorescence. The involvement of the low-affinity sites in the Ca2+-induced effect is consistent with the fact that Mg2+ has no effect on pyrene fluorescence. On rapid mixing of the pyr-TnI-TnC complex with Ca2+ in a stopped-flow apparatus, most of the excimer decrease is complete within the instrumental dead time, indicating a rate constant k greater than 350 s-1, which is comparable to that of the conformational change in TnC resulting from Ca2+ binding to the low-affinity sites. Rapid mixing of the Mg2-TnC-pyr-TnI complex with Ca2+ yields similar results, suggesting that the type of metal ion present at the high-affinity sites has little, if any, effect on the probe. It has been suggested previously that Cys 48 and 64 are located in a TnT-binding region of TnI (Chong P.C.S. and Hodges, R.S. (1982) J. Biol. Chem. 255, 3757). Our results suggest that a Ca2+-induced structural change in the TnI-binding region of TnC could be transmitted to TnT by affecting the TnT-binding region of TnI as part of the chain of events in the regulation of muscle contraction.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D009132:Muscles / Q000378:metabolism*; D011487:Protein Conformation; D011721:Pyrenes; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D011721:Pyrenes; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; C030984:pyrene; D002118:Calcium