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

Abstract

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 (YlO in the N-helix, and Y109 in site 3 in the C-terminal domain) in distinct microenviromnents: 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 (Cat'-free) and Ca*'-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 Fao&, but interestingly the Ca2' effect is completely suppressed (AF = Fe, -&.xA = 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'+; whereas Y 10 in the N-helix is dominant in Ca*'-free solution, Y 109 is the sole contributor to the Ca*+ effect. Furthermore, to explain the biphasic fluorescence response of Y 109 obtained during Ca*+ titrations, the hndings yield the most unequivocal evidence that Ca*+-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.

Functional Role of Arginine-11 in the N-Terminal Helix of Skeletal Troponin C: Combined Mutagenesis and Molecular Dynamics Investigation

“…In contrast to CaM, the function of TnC as an on/off switch for muscular contraction seems to depend on the precise separation between the two lobes during the complexation of TnC with Tnl (Babu et al, 1993;Keleti et al, 1994;Olah & Trewhella, 1994). Therefore, the present comparative analysis of functional and molecular properties of WT sTnC and its variants centers on the properties that are likely to determine the differences between TnC and CaM in their mechanisms of interaction with targets.…”