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Disparate contributions of Tyr 10 and Tyr 109 to fluorescence intensity of rabbit skeletal muscle troponin C identified using a genetically engineered mutant
Author(s) -
Keleti David,
Rao Venu G.,
Su Hong,
Akella Arvind B.,
Ding Xiao-Ling,
Gulati Jagdish
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)01095-1
Subject(s) - egta , fluorescence , troponin c , chemistry , biophysics , mutant , calcium , helix (gastropod) , biochemistry , troponin , biology , quantum mechanics , psychiatry , myocardial infarction , gene , snail , psychology , ecology , physics , organic chemistry
Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca 2+ ‐induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (Y10 in the N‐helix, and Y109 in site 3 in the C‐terminal domain) in distinct microenvironments: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady‐state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Ca 2+ ‐free) and Ca 2+ ‐saturated (pCa4) solutions. For the wild‐type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild‐type F EGTA ), but interestingly the Ca 2+ effect is completely suppressed (Δ F = F pCa4 ‐ F EGTA = 2% of the wild‐type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild‐type sTnC, both in the presence and absence of Ca 2+ ; whereas Y10 in the N‐helix is dominant in Ca 2+ ‐free solution, Y109 is the sole contributor to the Ca 2+ effect. Furthermore, to explain the biphasic fluorescence response of Y109 obtained during Ca 2+ titrations, the findings yield the most unequivocal evidence that Ca 2+ ‐induced conformational changes in the trigger sites operating the contractile switch modify properties of the C‐terminal sites in TnC pari passu.