Amazing stuff!
"We are, quite literally, held together by collagen. Structured like a twisted rope, the protein accounts for 15% to 20% of the protein in our bodies; it plays an essential role in mechanically supporting our cells and tissues. But for the past two decades, researchers have been puzzling over a biological riddle: Collagen is inherently unstable. At our own body temperature, it falls apart like a frayed rope. Now, results recently detailed at a major physics conference have revealed one of the key ways that collagen holds its form: clusters of molecular “staples.” ...
To understand exactly how these triple helices fall apart and come back together, researchers ... painstakingly imaged hundreds of them as they unraveled at different temperatures. Doing so let the researchers determine that clusters of disulfide bonds (which the researchers call “cysteine knots”) act as structural staples, stabilizing collagen’s twisted form. ...
the researchers also found that DNA sequences that encode for cysteine knots can be found in everything from jellyfish to mammals. Evidently, cysteine knots abound on the tree of multicellular life—supporting collagen as it supports us all"
"Synopsis
Collagen emerged at the dawn of multicellular life as an essential building block of tissues. Our understanding has evolved from viewing collagen as merely a physical framework to recognizing it as a dynamic scaffold that regulates cellular responses through mechanotransduction.
Intriguingly, collagen assembles into higher-order structures that provide mechanical support to tissues, despite being thermally unstable at body temperature.
However, studies on collagen’s sequence, structure, and mechanics have primarily relied on short model peptides, which often fail to accurately represent native collagen sequences. Hence, there is a growing need for the development of new methods that enable the study of native collagen in its full-length context, offering insights into how it upholds its dynamic function despite its thermal instability. Using atomic force microscopy (AFM) imaging, I investigated collagen’s response to temperature and observed a time-dependent loss of folded protein at body temperature, including an overall shortening of the contour length, reflecting structural destabilization. I further characterized the bending stiffness profile of collagen IV as a function of temperature and identified a putative initiation site for thermally induced unfolding.
My findings also revealed that interchain disulfide bonds enhance the thermal stability of collagen IV and serve as the primary nucleation site for in vitro refolding.
Additionally, multiple sequence alignments across diverse species uncovered an evolutionarily conserved cystine knot present across metazoan phyla, underscoring its significance in early collagen IV structures. These results provide new insights into collagen’s thermal response and the first steps to establishing relations between its sequence-encoded information and mechanical properties in these large proteins."
From the abstract:
"Collagen has been evolutionarily selected as the preferred building block of extracellular structures. Despite the inherent and surprising 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 response of collagen types I, III, and particularly IV to changes in temperature.
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."
Molecular ‘staples’ resolve puzzle of how collagen stays together "Research reveals how the key structural protein, inherently unstable at body temperature, forms resilient twists"
Decoding Collagen’s Unfolding and Refolding Pathways with AFM Imaging (original news release)
BPS2025 - Decoding collagen's thermally induced unfolding and refolding pathways using AFM imaging (no public access)
Decoding Collagen’s Thermally Induced Unfolding and Refolding Pathways (preprint, open access)
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