Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2626 HZ Delft, The Netherlands.
Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2626 HZ Delft, The Netherlands.
J Inorg Biochem. 2018 Jul;184:42-49. doi: 10.1016/j.jinorgbio.2018.04.011. Epub 2018 Apr 11.
The study of the structure, function, folding and conformational transitions of cytochrome c is of great interest because this protein plays an important role in biological electron transport and apoptosis. The different native and non-native conformations have been studied extensively under equilibrium conditions at different pH values, however, kinetic studies are rare because they require technically challenging rapid mixing and spectroscopic monitoring techniques. Here we present the refolding kinetics of acid denatured cytochrome c using the pH jump technique from pH 2 to pH 4.7 in combination with a new ultrafast continuous flow mixing device that allows time resolved measurements to the microsecond time scale. Our results show that the initial refolding of denatured oxidized cytochrome c occurs very rapidly with a time constant τ = 10 μs, and is followed by discrete refolding steps with time constants of 56 and 208 μs. Electron paramagnetic resonance analysis of the different intermediates, obtained by microsecond freeze hyper quenching showed that the first two intermediates are predominantly high spin, and the third intermediate is the low spin species with complete His/Met coordination. The initial rapid phase is characterized by the formation of high spin species distinct from the completely unfolded state. We interpret this as the formation of a five coordinate species with His18 as the axial ligand or six coordinate with water and His18 as the axial ligands.
研究细胞色素 c 的结构、功能、折叠和构象转变具有重要意义，因为这种蛋白质在生物电子传递和细胞凋亡中起着重要作用。已经在不同 pH 值下的平衡条件下广泛研究了不同的天然和非天然构象，但动力学研究很少，因为它们需要技术上具有挑战性的快速混合和光谱监测技术。在这里，我们使用 pH 跃变技术从 pH 2 到 pH 4.7 研究了酸变性细胞色素 c 的重折叠动力学，该技术结合了一种新的超快连续流动混合装置，允许在微秒时间尺度上进行时间分辨测量。我们的结果表明，变性氧化细胞色素 c 的初始重折叠非常迅速，时间常数 τ=10 μs，随后是离散的重折叠步骤，时间常数为 56 和 208 μs。通过微秒冷冻超快速淬火获得的不同中间体的电子顺磁共振分析表明，前两个中间体主要是高自旋，第三个中间体是低自旋物种，具有完整的 His/Met 配位。初始快速相的特征是形成不同于完全展开状态的高自旋物种。我们将其解释为形成具有 His18 作为轴向配体的五配位物种或具有水和 His18 作为轴向配体的六配位物种。
