Ca2+ dependence of the distance between Cys-98 of troponin C and Cys-133 of troponin I in the ternary troponin complex. Resonance energy transfer measurements.
We have used resonance energy transfer to study the spatial relationship between Cys-98 of rabbit skeletal troponin C and Cys-133 of rabbit skeletal troponin I in the reconstituted ternary troponin complex. The donor was introduced by labeling either troponin C or troponin I with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, while the acceptor was introduced by labeling either protein with N-[4-(dimethylamino)phenyl-4'-azophenyl]maleimide. The extent of energy transfer was determined by measuring the quenching of the donor fluorescence decay. The results indicate first that the distance between these two sites is not fixed, suggesting that the protein regions involved possess considerable segmental flexibility. Second, the mean distance between the two sites is dependent on the metal-binding state of troponin C, being 39.1 A when none of the metal-binding sites are occupied, 41.0 A when Mg2+ ions bind at the high-affinity sites, and 35.5 A when Ca2+ ions bind to the low-affinity sites. Neither the magnitude of the distances nor the trend of change with metal ions differs greatly when the locations of the probes are switched or when steady-state fluorometry was used to determine the transfer efficiency. Since the low-affinity sites have been implicated as the physiological triggering sites, our findings suggest that one of the key events in Ca2+ activation of skeletal muscle contraction is a approximately 5-A decrease in the distance between the Cys-98 region of troponin C and the Cys-133 region of troponin I.
Department of Muscle Research, Boston Biomedical Research Institute, Harvard Medical School, Massachusetts 02114.
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Tao
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Gowell
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Strasburg
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Gergely
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Leavis
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003545:Cysteine; D004735:Energy Transfer; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D011487:Protein Conformation; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I
D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D003545:Cysteine; D002118:Calcium