Time-resolved circular dichroism studies of protein folding intermediates of cytochrome c
Time-resolved circular dichroism studies of protein folding intermediates of cytochrome c
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DOI：
10.1021/bi972369f
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发表时间：
1998-04-21
期刊：
影响因子：
2.9
通讯作者：
Kliger, DS
中科院分区：
文献类型：
--
作者：
Chen, EF;Wood, MJ;Kliger, DS
细胞色素c （cytc）在4.6 M盐酸胍（pH 6.5）中的圆二色性光谱表明，还原的cytc的二级结构接近原生结构，而在氧化石墨烯结合的物种（COCytc）中，二级结构基本展开。因此，COCytc的光解会引起二级结构的巨大变化，这可以用远紫外区时间分辨圆二色性（TRCD）光谱来探测。利用时间分辨吸收（TROA）和TRCD方法研究COCytc的光解反应，在纳秒到秒的时间尺度上鉴定细胞折叠的结构中间体。来自Soret地区的TROA数据与之前的研究相似，显示了四种中间体的寿命分别为2,50,225和880 μ s。2 μ s的过程可能涉及Fe(II)-Met80配位。在500 ns内220 nm的TRCD信号中观察到大约7%的原生CD信号，没有观察到明显的额外二级结构形成。2 μ s后进一步折叠可能被His26/His33与Fe（II）的结扎所抑制，这可能与50 μ s相有关。在混合气体实验的基础上，tau = 225和880 μ s这两个最慢的组分归因于CO的再结合。CO重结合有望与蛋白质折叠竞争，并有利于未折叠状态。然而，当通过降低CO浓度将两个CO重结合寿命延长到毫秒级时，在220 nm处CD信号仍然没有显著增加。
The circular dichroism spectra of cytochrome c (cytc) in 4.6 M guanidine hydrochloride (pH 6.5) indicate that the secondary structure in reduced cytc is near-native, whereas in the GO-bound species (COCytc) it is substantially unfolded. Photolysis of COCytc should thus induce large changes in the secondary structure, which can be probed with time-resolved circular dichroism (TRCD) spectroscopy in the far-UV region. Time-resolved absorption (TROA) and TRCD methods were used to study the photolysis reaction of COCytc in efforts to identify structural intermediates in cytc folding on time scales from nanoseconds to seconds. TROA data from the Soret region, similar to previous studies, showed four intermediates with lifetimes of 2, 50, 225, and 880 mu s. The 2-mu s process is proposed to involve Fe(II)-Met80 coordination. Approximately 7% of the native CD signal was observed in the TRCD signal at 220 nm within 500 ns, with no significant additional secondary structure formation observed. Further folding after 2 mu s may be inhibited by ligation of His26/His33 with Fe(II), which is suggested to be associated with the 50-mu s phase. The two slowest components, tau = 225 and 880 mu s, are attributed to CO rebinding on the basis of mixed-gas experiments. CO rebinding is expected to compete with protein folding and favor the unfolded state. However, when the two CO rebinding lifetimes are extended into milliseconds by reducing the CO concentration, there is still no significant increase in CD signal at 220 nm.