Nanosecond dynamics of horse heart apocytochrome c in aqueous solution as studied by time-resolved fluorescence of the single tryptophan residue (Trp-59).
Nanosecond dynamics of horse heart apocytochrome c in aqueous solution as studied by time-resolved fluorescence of the single tryptophan residue (Trp-59).
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通过单个色氨酸残基 (Trp-59) 的时间分辨荧光研究马心脱辅细胞色素 c 在水溶液中的纳秒动力学。
DOI：
10.1021/bi00424a010
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
J. Gallay
中科院分区：
文献类型：
--
作者：
M. Vincent;J. Brochon;F. Mérola;W. Jordi;J. Gallay
研究了线粒体内细胞色素c的前体--马心脱辅基细胞色素c的单个色氨酸残基（Trp-59）在水溶液中的时间分辨荧光发射特性。通过非线性最小二乘法将在整个发射光谱上测量的总荧光强度衰减分析为三个或四个指数的总和，最后一个模型总是提供卡方值的轻微但显著的降低。最近开发的用于时间分辨荧光测定的最大熵分析（Livesey等人，1987; Livesey & Brochon，1987），强烈建议存在一个分布，至少包括四个独立的类的寿命。中心值为0.1 - 0.2，1，3，和5纳秒左右，在协议的非线性最小二乘回归分析得到的寿命值。作为发射波长的函数，这些值在实验误差内保持恒定，而分数振幅的重新分布被观察到：短分量的贡献在发射光谱的蓝色边缘区域增加。温度升高导致分数振幅的重新分布，主要影响5 ns分量，在高温（35 - 40 ℃）下几乎完全消失。的寿命值没有显着影响，除了3-ns的组件，这减少了约15%的温度范围内研究。这样的观察结果强烈表明，蛋白质存在于不同的构象亚状态下的热平衡。时间分辨荧光各向异性测量证明了快速内旋转的色氨酸残基的存在。计算出平均最大受限旋转角度约为55度。在各向异性衰减曲线上检测到第二内部运动，慢1个数量级，可能对应于涉及Trp-59残基的肽链的局部运动。最后，最长的相关时间（5 ns）应对应于整个蛋白质的平均旋转。其值作为蛋白质浓度的函数加倍，揭示了在所研究的浓度范围（2 - 139 μ M）中最可能导致二聚体的缔合过程。肽链的柔性在缔合形式中比在单体形式中更受限制，但色氨酸残基的快速内旋转不是。
The time-resolved fluorescence emission characteristics of the single tryptophan residue (Trp-59) of horse heart apocytochrome c--the precursor of the intramitochondrial cytochrome c--were studied in aqueous solution. The total fluorescence intensity decay measured over the whole emission spectrum was analyzed as a sum of three or four exponentials by the nonlinear least-squares method, the last model always providing a slight but significant decrease in the chi 2 values. Maximum entropy analysis, recently developed for time-resolved fluorometry (Livesey et al., 1987; Livesey & Brochon, 1987), strongly suggests the existence of a distribution including at least four separate classes of lifetimes. The center values were around 0.1-0.2, 1, 3, and 5 ns, in agreement with the lifetime values obtained by nonlinear least-squares regression analysis. As a function of the emission wavelength, these values remained constant within the experimental error, whereas a redistribution of the fractional amplitudes was observed: the contributions of the short components increased in the blue edge region of the emission spectrum. Temperature increase led essentially to a redistribution of the fractional amplitudes, affecting mostly that of the 5-ns component, which almost totally disappeared at high temperature (35-40 degrees C). The lifetime values were not significantly affected except for the 3-ns component, which decreased by about 15% in the temperature range studied. Such observations strongly suggest that the protein exists under different conformational substates in thermal equilibrium. Time-resolved fluorescence anisotropy measurements evidenced the existence of fast internal rotation of the Trp residue. An average maximum restricted angle of rotation of around 55 degrees was calculated. A second internal motion, slower by 1 order of magnitude, corresponding likely to a local motion of the peptide chain involving the Trp-59 residue, was detected on the anisotropy decay curve. Finally, the longest correlation time (5 ns) should correspond to the average rotation of the overall protein. Its value doubled as a function of the protein concentration, revealing an association process leading most likely to a dimer in the concentration range studied (2-139 microM). The flexibility of the peptide chain was more restrained in the associated than in the monomeric form, but the fast internal rotation of the Trp residue was not.
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影响因子：
2.9
作者：
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DOI：
--
发表时间：
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期刊：
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影响因子：
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作者：
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影响因子：
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