Folding of collagen IV

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°C with a midpoint at 37°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°C following complete unfolding at 50°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 es