The predominant structural protein in vertebrates is collagen, which plays a key role in extracellular matrix and connective tissue mechanics. Despite its prevalence and physical importance in biology, the mechanical properties of molecular collagen are far from established. The flexibility of its triple helix is unresolved, with descriptions from different experimental techniques ranging from flexible to semirigid. Furthermore, it is unknown how collagen type (homo-versus heterotrimeric) and source (tissue derived versus recombinant) influence flexibility. Using SmarTrace, a chain-tracing algorithm we devised, we performed statistical analysis of collagen conformations collected with atomic force microscopy to determine the protein's mechanical properties. Our results show that types I, II, and III collagens-the key fibrillar varieties-exhibit similar molecular flexibilities. However, collagen conformations are strongly modulated by salt, transitioning from compact to extended as KCl concentration increases in both neutral and acidic pH. Although analysis with a standard worm-like chain model suggests that the persistence length of collagen can attain a wide range of values within the literature range, closer inspection reveals that this modulation of collagen's conformational behavior is not due to changes in flexibility but rather arises from the induction of curvature (either intrinsic or induced by interactions with the mica surface). By modifying standard polymer theory to include innate curvature, we show that collagen behaves as an equilibrated curved worm-like chain in two dimensions. Analysis within the curved worm-like chain model shows that collagen's curvature depends strongly on pH and salt, whereas its persistence length does not. Thus, we find that triple-helical collagen is well described as semiflexible irrespective of source, type, pH, and salt environment. These results demonstrate that collagen is more flexible than its conventional description as a rigid rod, which may have implications for its cellular processing and secretion.

The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5′ end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5′ end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.

Despite its prevalence and physical importance in biology, the mechanical properties of molecular collagen are far from established. The flexibility of the triple helix is unresolved, with descriptions from different experimental techniques ranging from flexible to semirigid. Furthermore, it is unknown how collagen type (homo-vs. heterotrimeric) and source (tissue-derived vs. recombinant) influence flexibility. Using SmarTrace, a chain tracing algorithm we devised, we performed statistical analysis of collagen conformations collected with atomic force microscopy (AFM) to determine the protein's mechanical properties. Our results show that types I, II and III collagens -the key fibrillar varietiesexhibit molecular flexibilities that are very similar. However, collagen conformations are strongly modulated by salt, transitioning from compact to extended as KCl concentration increases, in both neutral and acidic pH. While analysis with a standard worm-like chain model suggests that the persistence length of collagen can attain almost any value within the literature range, closer inspection reveals that this modulation of collagen's conformational behaviour is not due to changes in flexibility, but rather arises from the induction of curvature (either intrinsic or induced by interactions with the mica surface). By modifying standard polymer theory to include innate curvature, we show that collagen behaves as an equilibrated curved worm-like chain (cWLC) in two dimensions. Analysis within the cWLC model shows that collagen's curvature depends strongly on pH and salt, while its persistence length does not. These results show that triple-helical collagen is well described as semiflexible, irrespective of source, type, pH and salt environment. SIGNIFICANCE STATEMENTThe predominant structural protein in vertebrates is collagen, which plays a key role in extracellular matrix and connective tissue mechanics. Previous measurements on molecular collagen have provided measures of flexibility that vary by over an order of magnitude. Thus, the mechanics of triple-helical collagen -the fundamental building block of structural tissues -are not established. Our results, obtained using single-molecule imaging analysis, find that collagen deposited on a mica surface adopts intrinsically curved structures at low salt and pH. After accounting for this curvature, we find that the flexibility of collagen (as determined by its persistence length) is not affected significantly by salt, pH or composition. Thus, collagen appears to be well described as a semiflexible polymer.

Extracellular matrix mechanics influence diverse cellular functions, yet surprisingly little is known about the mechanical properties of their constituent collagens. In particular, network-forming collagen IV, an integral component of basement membranes, has been far less studied than fibril-forming collagens. A key feature differentiating collagen IV is the presence of interruptions in the triple-helix-defining (Gly-X-Y) sequence along its collagenous domain. Here, we used atomic force microscopy (AFM) to determine the impact of these interruptions on the flexibility of collagen. Our extracted flexibility profile reveals that collagen IV possesses highly heterogeneous mechanics, ranging from semi-flexible regions as found for fibril-forming collagens to a lengthy region of high flexibility towards its N terminus. A simple model in which flexibility is dictated only by the presence of interruptions fit the extracted profile reasonably well, providing insight into the alignment of chains and supporting the role of interruptions in instilling flexibility. However, limitations of this model were illuminated by our determination of variable flexibility along continuously triple-helical collagen III, which we found to possess a high-flexibility region around its matrix-metalloprotease (MMP) binding site. Surprisingly, proline content did not correlate with local flexibility in either collagen type. We also found that physiologically relevant changes in pH and chloride concentration did not alter the flexibility of collagen IV, indicating such environmental changes are not used to control its compaction during secretion. Although extracellular chloride ions play a role in triggering collagen IV network formation, they do not appear to modulate the structure of its collagenous domain.

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