FOLDING OF COLLAGEN-IV
FOLDING OF COLLAGEN-IV
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DOI：
10.1111/j.1432-1033.1988.tb14458.x
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发表时间：
1988-12-15
期刊：
影响因子：
--
通讯作者：
KUHN, K
中科院分区：
文献类型：
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作者：
DOLZ, R;ENGEL, J;KUHN, K
通过用细菌胶原酶有限消化从小鼠Engelbreth-Holm-Swarm（EHS）肉瘤组织分离由其C-末端球状NC 1结构域连接的两个胶原IV分子的胶原IV二聚体。与400 nm的完整胶原IV相比，胶原结构域仅为300 nm长，但保留了主要三螺旋的N-末端区域中的二硫键。通过圆二色性监测，胶原结构域的解折叠发生在30 - 44 ℃的温度范围内，中点在37 ℃。该过渡比胶原蛋白I、II和III中的连续三螺旋的过渡明显更宽，这一特征可以通过胶原蛋白IV的三螺旋结构域中的频繁非胶原中断来解释。在50 ℃完全解折叠后在25 ℃的重折叠通过圆二色性、非重折叠片段的选择性蛋白水解消化和通过新开发的方法监测，其中通过电子显微镜观察回收的三螺旋片段。三螺旋的形成被发现从C-末端NC 1结构域向N-末端以拉链样的方式进行，这表明该结构域对于成核是必不可少的。对于具有完整NC 1结构域的胶原IV二聚体，三螺旋生长的速率与胶原III的速率相当，这表明非胶原中断不会减慢重折叠过程，其中限速步骤是脯氨酸肽键的顺-反异构化。复性率接近100%，通过热稳定性和电子显微镜观察，复性产物与起始材料相似。通过胃蛋白酶去除NC 1结构域或通过乙酸解离其六元结构导致重折叠能力的丧失。相反，在缓慢反应中形成具有随机分散的短三螺旋片段的产物。在任何情况下，即使三螺旋结构域的N-末端区域中的二硫键是完整的，也没有观察到从N-向C-末端的重折叠。与其他胶原蛋白的结果一起，这表明C至N方向性是三螺旋折叠的固有性质。
Collagen IV dimers of two collagen IV molecules connected by their C-terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth-Holm-Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N-terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44.degree.C with amidpoint at 37.degree.C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non-collagenous interruptions in the triple-helical domain of collagen IV. Refolding at 25.degree.C following complete unfolding at 50.degree.C was monitored by circular dichroism, selective proteolytic digestion of non-refolded segments and by a newly developed method in which the recovered triple-helical segments were visualized by electron microscopy. Triple-helix formation was found to proceed in a zipper-like fashion from the C-terminal NC1 domains towards the N-terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple-helix growth was of comparable magnitude to that of collagen III, demonstrating that the non-collagenous interruptions do not slow down the refolding process where the rate-limiting step is the cis-trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly disperse short triple-helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N-terminal region of the triple-helical domain were intact, was refolding from the N- towards the C-terminus observed. Taken together with results in other collagens, this suggests that C to N directionality right be an intrinsic property of triple-helix folding.