The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine-substitution of the aspartate residue in IS3 disturbed the intra-loop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments.

中文翻译：

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插入环介导的折叠传播控制高温下超嗜热 Tk-枯草杆菌蛋白酶的有效成熟

与其嗜温对应物相比，来自超嗜热古菌 Thermococcus kodakarensis 的丝氨酸蛋白酶 Tk-枯草杆菌蛋白酶在其表面具有三个插入环 (IS1-IS3)。尽管 Tk-枯草杆菌蛋白酶在高温下成熟需要 IS1 和 IS2，但 IS3 的作用仍然未知。在这里，CD 光谱显示 IS3 缺失在中间状态阻止了 Tk-枯草杆菌蛋白酶折叠，其中形成了中央核，但没有发生随后折叠传播到末端亚域。IS3 中天冬氨酸残基的丙氨酸取代扰乱了环内氢键网络，正如晶体学分析所证明的那样，导致高温下折叠受损。考虑到 IS3 在超嗜热同源物中的高度保守性，