Disparate contributions of Tyr10 and Tyr109 to fluorescence intensity of rabbit skeletal muscle troponin C identified using a genetically engineered mutant.
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 FEGTA), but interestingly the Ca2+ effect is completely suppressed (delta F = FpCa4-FEGTA = 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 Ca2+; whereas Y10 in the N-helix is dominant in Ca(2+)-free solution, Y109 is the sole contributor to the Ca2+ effect. Furthermore, to explain the biphasic fluorescence response of Y109 obtained during Ca2+ 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.
Keleti
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D
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461.
Rao
V G
VG
Su
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H
Akella
A B
AB
Ding
X L
XL
Gulati
J
J
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005453:Fluorescence; D018482:Muscle, Skeletal / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014336:Troponin / Q000737:chemistry* / Q000235:genetics; D019209:Troponin C; D014443:Tyrosine / Q000737:chemistry*
D014336:Troponin; D019209:Troponin C; D014443:Tyrosine; D004533:Egtazic Acid; D002118:Calcium