AN EARLY IMMUNOREACTIVE FOLDING INTERMEDIATE OF THE TRYPTOPHAN SYNTHASE-BETA-2 SUBUNIT IS A MOLTEN GLOBULE
AN EARLY IMMUNOREACTIVE FOLDING INTERMEDIATE OF THE TRYPTOPHAN SYNTHASE-BETA-2 SUBUNIT IS A MOLTEN GLOBULE
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DOI：
10.1016/0014-5793(90)80703-l
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发表时间：
1990-04-09
期刊：
影响因子：
3.5
通讯作者：
SUGAI, S
中科院分区：
文献类型：
--
作者：
GOLDBERG, ME;SEMISOTNOV, GV;SUGAI, S
使用停流技术，通过圆二色性 (CD) 和荧光疏水探针 (ANS) 的结合研究了色氨酸合酶 β 2 亚基的重折叠动力学。恢复天然远紫外 CD 信号的动力学表明，在尿素变性 β2 重折叠时，超过一半的蛋白质二级结构在 CD 停流装置的死区时间内（0.013 s）形成。另一方面，当胍未折叠β 2 重新折叠时，ANS 的荧光在大约 1 秒后达到最大值，然后“缓慢”减少。这些结果表明，早期瞬时折叠中间体在 1-10 秒的时间范围内积累，该中间体具有明显的二级结构和对 ANS 的高亲和力。在此时间范围内，近紫外 CD 仍然很低。因此，这种瞬态中间体似乎具有“熔球”态的所有特征 [(1987) FEBS Lett. 224, 9-13]。此外，通过比较这种瞬时中间体消失的固有时间（t 1 2 35 s）与先前表征的[(1988) Biochemistry 27, 7633-7640]单克隆抗体识别的早期免疫反应中间体的形成时间（t 1 2 12 s），结果表明，在蛋白质紧密堆积之前，这种天然样表位在“熔球”内形成侧链。
The refolding kinetics of the tryptophan synthase β 2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured β 2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded β 2 the fluorescence of ANS passes through a maximum after about 1 s and then ‘slowly’decreases. These results show the accumulation, in the 1–10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the ‘molten globule’state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t 1 2 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early imuno-reactive intermediate recognized by a monoclonal antibody (t 1 2 12 s), it is shown that this native-like epitope forms within the ‘molten globule’, before the tight packing of the protein side chains.