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bioRxiv · 10.1101/2024.10.02.616390

Decoding Collagen's Thermally Induced Unfolding and Refolding Pathways

Al-Shaer, A.·Forde, N. R.
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Abstract

Collagen has been evolutionarily selected as the preferred building block of extracellular structures. Despite inherent thermal instability of individual proteins at body temperature, collagen manages to assemble into higher-order structures that provide mechanical support to tissues. Sequence features that enhance collagen stability have been deduced largely from studies of collagen-mimetic peptides, as the large sizes of collagens have precluded high-resolution studies of their structure. Thus, there is a need for new methods to analyze the structure and mechanics of native collagen proteins. In this study, we used AFM imaging to investigate the temperature response of collagen types I, III and particularly IV. We observed a time-dependent loss of folded structures upon exposure to body temperature, with structural destabilization along the collagenous domain reflected by a shorter overall contour length. We characterized the sequence-dependent bending stiffness profile of collagen IV as a function of temperature and identified a putative initiation site for thermally induced unfolding. Interchain disulfide bonds in collagen IV were shown to enhance thermal stability and serve as the primary nucleation sites for in vitro refolding. In contrast to the canonical C-to-N terminal folding direction, we found an interchain cystine knot to enable folding in the opposite direction. Multiple sequence alignments revealed that this cystine knot is evolutionarily conserved across metazoan phyla, highlighting its significance in the stabilization of early collagen IV structures. Our findings provide mechanistic insights into the unfolding and refolding pathway of collagen IV, providing valuable insight into how its heterogeneous sequence influences stability and mechanics.

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Al-Shaer, A., Forde, N. R.. 2024-10-03. Decoding Collagen's Thermally Induced Unfolding and Refolding Pathways. https://doi.org/10.1101/2024.10.02.616390

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