Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils.
Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils.
复制标题
芘标记的心肌肌钙蛋白 C。Ca2 对溶液和肌原纤维中单体和准分子荧光的影响。
DOI：
10.1016/s0006-3495(92)81896-9
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发表时间：
1992
影响因子：
3.4
通讯作者：
Fuchs,F
中科院分区：
文献类型：
--
作者：
Liou,YM;Fuchs,F
用含芘的SH-反应性化合物N-（1-芘）马来酰亚胺和N-（1-芘）碘乙酰胺标记牛心肌肌钙蛋白C（cTnC）的两个半胱氨酸残基（Cys-35和Cys-84），以研究cTnC调节结构域中与阳离子结合和跨桥连接相关的构象变化。标记的cTnC具有芘的特征荧光光谱，具有两个尖锐的单体荧光峰和一个宽的准分子荧光峰。激基缔合物荧光是由相邻芘基团的二聚化引起的。金属（Mg 2+或Ca 2+）与cTnC的高亲和力位点（位点III和IV）结合时，单体荧光略有降低，但对激基缔合物荧光无影响。与此相反，钙离子结合到低亲和力的监管（网站II）网站elechtan增加单体荧光和减少准分子荧光。这些结果可以解释为，假设芘连接到Cys-84被拉到一个疏水性的口袋中形成的结合的Ca 2+的网站II。当标记的cTnC掺入肌钙蛋白复合物或取代到心肌肌原纤维中时，单体荧光增强，而准分子荧光降低。这表明，与其他监管组件在细丝的关联可能会影响的接近度（或流动性）的两个芘基团的方式类似的钙离子结合。随着Ca 2+与位点II的结合，准分子荧光进一步减弱，而单体荧光没有显着变化。在肌原纤维中，跨桥分离（5 mM MgATP，pCa 8.0）导致单体荧光减少，但对受激准分子荧光没有影响。然而，饱和的cTnC与Ca 2+减少激基缔合物荧光，但不会导致单体荧光的进一步变化。因此，芘荧光光谱定义了与弱结合、循环和严格交叉桥相关的cTnC的不同构象。
The two cysteine residues (Cys-35 and Cys-84) of bovine cardiac troponin C (cTnC) were labeled with the pyrene-containing SH-reactive compounds, N-(1-pyrene) maleimide, and N-(1-pyrene)iodoacetamide in order to study conformational changes in the regulatory domain of cTnC associated with cation binding and cross-bridge attachment. The labeled cTnC exhibits the characteristic fluorescence spectrum of pyrene with two sharp monomer fluorescence peaks and one broad excimer fluorescence peak. The excimer fluorescence results from dimerization of adjacent pyrene groups. With metal binding (Mg2+ or Ca2+) to the high affinity sites of cTnC (sites III and IV), there is a small decrease in monomer fluorescence but no effect on excimer fluorescence. In contrast, Ca2+ binding to the low affinity regulatory (site II) site elicits an increase in monomer fluorescence and a reduction in excimer fluorescence. These results can be accounted for by assuming that the pyrene attached to Cys-84 is drawn into a hydrophobic pocket formed by the binding of Ca2+ to site II. When the labeled cTnC is incorporated into the troponin complex or substituted into cardiac myofibrils the monomer fluorescence is enhanced while the excimer fluorescence is reduced. This suggests that the association with other regulatory components in the thin filament might influence the proximity (or mobility) of the two pyrene groups in a way similar to that of Ca2+ binding. With the binding of Ca2+ to site II the excimer fluorescence is further reduced while the monomer fluorescence is not changed significantly. In myofibrils, cross-bridge detachment (5 mM MgATP, pCa 8.0) causes a reduction in monomer fluorescence but has no effect on excimer fluorescence. However, saturation of the cTnC with Ca2+ reduces excimer fluorescence but causes no further change in monomer fluorescence. Thus, the pyrene fluorescence spectra define the different conformations of cTnC associated with weak-binding, cycling, and rigor cross-bridges.