Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C.
Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C.
复制标题
工程骨骼肌肌钙蛋白 C 的单一色氨酸的时间分辨荧光研究。
DOI：
10.1016/s0006-3495(97)78137-2
复制
发表时间：
1997
影响因子：
3.4
通讯作者：
Cheung,HC
中科院分区：
文献类型：
--
作者：
She,M;Dong,WJ;Umeda,PK;Cheung,HC
利用基因突变体研究了鸡骨骼肌肌钙蛋白C（TnC）的调节结构域，该突变体在22、52和90位含有单一色氨酸。在Mg ~（2+）（2-Mg态）和Ca ~（2+）（4-Ca态）存在下，Trp-22的量子产率分别为0.33和0.25。2-Mg态的大量子产率是由于相对小的非辐射衰减速率，并且与331 nm处的发射峰一致。这种状态的强度衰减是单指数的，单寿命为5.65 ns，与波长无关。在4-Ca状态下，衰减是双指数的两个寿命的平均值从4.54增加到4.92 ns的整个发射带。短寿命的衰变相关光谱相对于稳态光谱红移了19 nm。Trp-52在2-Mg状态下呈双指数衰减，在4-Ca状态下呈三指数衰减。Trp-90的衰减需要三个指数项才能令人满意地拟合，但在4-Ca状态下可以用两个指数项拟合。这两种色氨酸的较低量子产率（< 0.15）是由于较小的辐射衰变率和较大的非辐射衰变率的结合。Trp-22的结果表明，在没有结合的Ca 2+的情况下，在调节位点和激活剂Ca 2+诱导的基态异质性的均匀基态吲哚环。Ca（2+）诱导的Trp-52和Trp-90的环境变化偏离了4-Ca状态的模型结构预测。所有三个的各向异性衰减的哈密顿量显示两个旋转相关时间。从Trp-22和Trp-90得到的长相关时间（phi 1=8.1-8.3 ns）表明不对称的流体动力学形状。TnC在结合激活剂Ca 2+后变得更加不对称（phi 1=10.1-11.6 ns）。从Trp-52得到的phi 1值比从Trp-22和Trp-90得到的值短3-4 ns，并且这些减少的相关时间可能与残基的移动性和/或局部片段柔性有关。
The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1=8.1–8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1=10.1–11.6 ns). The values of phi 1 obtained from Trp-52 are 3–4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.