Dissection of complex protein dynamics in human thioredoxin
Dissection of complex protein dynamics in human thioredoxin
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DOI：
10.1073/pnas.0608498104
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发表时间：
2007-03-27
影响因子：
11.1
通讯作者：
Zhong, Dongping
中科院分区：
文献类型：
--
作者：
Qiu, Weihong;Wang, Lijuan;Zhong, Dongping
我们通过剖析基本过程并确定其相关的时间尺度，报告了对人类硫氧还蛋白中复杂蛋白质动力学的直接研究。通过将定点诱变与飞秒光谱相结合，我们区分了硫氧还蛋白活性位点的四个部分时间重叠的动力学过程。使用内在色氨酸作为分子探针并通过突变研究，我们确定了蛋白质侧链对溶剂化的贡献可以忽略不计，并观察到活性位点的水合动力学发生在 0.47-0.67 和 10.8-13.2 ps。在还原态和氧化态下，我们确定了三个突变体在 10-17.5 ps 内激发的色氨酸和附近的二硫键之间的电子转移猝灭动力学。无论探针周围是否有相邻的带电、极性和疏水性残基，氧化还原态和所有突变体中都存在 95-114 ps 的稳健动力学过程，这归因于与其相邻肽键的电荷转移反应。定点突变还揭示了氢键距离为 275-615 ps 的天冬氨酸残基的电子猝灭动力学。通过测量各向异性随时间变化而确定的局部旋转动力学揭示了相对刚性的局部配置，但意味着蛋白质在超过纳秒的时间尺度上波动。这些结果阐明了水合运动、电子转移反应和活性位点局部蛋白质波动的时间演化，并显示了生物功能在广泛时间尺度上的持续协同动力学。
We report our direct study of complex protein dynamics in human thioredoxin by dissecting into elementary processes and determining their relevant time scales. By combining site-directed mutagenesis with femtosecond spectroscopy, we have distinguished four partly time-overlapped dynamical processes at the active site of thioredoxin. Using intrinsic tryptophan as a molecular probe and from mutation studies, we ascertained the negligible contribution to solvation by protein sidechains and observed that the hydration dynamics at the active site occur in 0.47-0.67 and 10.8-13.2 ps. With reduced and oxidized states, we determined the electron-transfer quenching dynamics between excited tryptophan and a nearby disulfide bond in 10-17.5 ps for three mutants. A robust dynamical process in 95-114 ps, present in both redox states and all mutants regardless of neighboring charged, polar, and hydrophobic residues around the probe, is attributed to the charge transfer reaction with its adjacent peptide bond. Site-directed mutations also revealed the electronic quenching dynamics by an aspartate residue at a hydrogen bond distance in 275-615 ps. The local rotational dynamics determined by the measurement of anisotropy changes with time unraveled a relatively rigid local configuration but implies that the protein fluctuates on the time scale of longer than nanoseconds. These results elucidate the temporal evolution of hydrating water motions, electron-transfer reactions, and local protein fluctuations at the active site, and show continuously synergistic dynamics of biological function over wide time scales.