Dong W J, Cheung H C
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294-2041, USA.
Biochim Biophys Acta. 1996 Jul 18;1295(2):139-46. doi: 10.1016/0167-4838(96)00028-3.
Residue Cys-84 of bovine cardiac troponin C (cTnC) located at the C-terminal end of helix D was selectively labeled in the presence of Ca2+ with two fluorescent probes: IAANS (2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) and acrylodan (6-acrylol-2-(dimethylamino)naphthalene). The fluorescence of the attached probes was studied by the steady-state and time-resolved methods to gain an insight about the nature of Ca(2+)-induced conformational changes in the N-domain regulatory region of cTnC. Changes in the experimental emission spectra, quantum yields, and excited-state lifetimes suggested that bound Ca2+ at the single regulatory site induced a less polar microenvironment for both probes attached to Cys-84. However, a twofold increase in the bimolecular collisional quenching constant was observed for both probes in the presence of activator Ca2+, indicating an increased exposure of the probes to solvent. These data were interpreted with reference to the origins of the observed Stokes' shifts. In the apo and 2Mg states of cTnC, the attached probes were partially shielded by helices B and C, and their excited-states were highly quenched in the tertiary structure through strong interactions of a dipolar nature with neighboring amino-acid side chains. In the 3Ca state, these interactions were disrupted so that nonradiative decay processes were suppressed and radiative processes were enhanced, leading to the observed increases in quantum yields and lifetimes and blue-shifts of the emission spectra. As the disruption of internal quenching resulted from separation of helices B and C from helix D, the attached probes became more accessible to solvent and experienced increases in the rate of collisions with external molecules in the solvent. Although this increased exposure to solvent would lead to suppression of radiative decay processes, this effect apparently was overcompensated by the effect of elimination of internal quenching. The present results are consistent with a Ca(2+)-induced open conformation of the N-domain in cTnC.
牛心肌肌钙蛋白C（cTnC）位于螺旋D C末端的半胱氨酸残基Cys-84在Ca2+存在的情况下用两种荧光探针进行了选择性标记：IAANS（2-（4-（碘乙酰胺基）苯胺基）萘-6-磺酸）和丙烯罗丹（6-丙烯酰-2-（二甲基氨基）萘）。通过稳态和时间分辨方法研究了附着探针的荧光，以深入了解cTnC N结构域调节区域中Ca(2+)诱导的构象变化的性质。实验发射光谱、量子产率和激发态寿命的变化表明，单个调节位点上结合的Ca2+为附着在Cys-84上的两种探针诱导了极性较小的微环境。然而，在激活剂Ca2+存在的情况下，两种探针的双分子碰撞猝灭常数都增加了两倍，这表明探针与溶剂的接触增加。这些数据参照观察到的斯托克斯位移的起源进行了解释。在cTnC的脱辅基和2Mg状态下，附着的探针被螺旋B和C部分屏蔽，并且它们的激发态在三级结构中通过与相邻氨基酸侧链的强偶极性质相互作用而高度猝灭。在3Ca状态下，这些相互作用被破坏，从而抑制了非辐射衰变过程并增强了辐射过程，导致观察到的量子产率和寿命增加以及发射光谱的蓝移。由于内部猝灭的破坏是由螺旋B和C与螺旋D分离导致的，附着的探针变得更容易与溶剂接触，并且与溶剂中外部分子的碰撞速率增加。尽管这种与溶剂接触的增加会导致辐射衰变过程受到抑制，但这种效应显然被消除内部猝灭的效应过度补偿了。目前的结果与cTnC中Ca(2+)诱导的N结构域开放构象一致。
