Disparate contributions of Tyr10 and Tyr109 to fluorescence intensity of rabbit skeletal muscle troponin C identified using a genetically engineered mutant

FEBS Letters

Su, Hong¹  Akella, Arvind B.¹  Rao, Venu G.¹  Gulati, Jagdish¹  Ding, Xiao-Ling¹  Keleti, David¹ 

[1] Molecular Physiology Laboratory, Division of Cardiology, Departments of Medicine and Physiology/Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461 USA

关键词: Troponin;    Calcium;    EF hand structure;    Muscle;    Mutagenesis;    Fluorescence;   

DOI  :  10.1016/0014-5793(94)01095-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca²⁺-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 (Ca²⁺-free) and Ca²⁺-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 Ca²⁺ effect is completely suppressed (ΔF = 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 Ca²⁺; whereas Y10 in the N-helix is dominant in Ca²⁺-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 Ca²⁺-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.

【 授权许可】

Unknown