Sequence-dependent mechanics of collagen reflect its structural and functional organization

Abstract

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.

AutoSmarTrace: Automated Chain Tracing and Flexibility Analysis of Biological Filaments

“…To assess performance when tracing experimental images, we compared estimates of persistence lengths of collagen proteins via manual and automated implementations of SmarTrace (6, 7), and also benchmarked by comparing to estimates of DNA’s persistence length on images recorded and analysed by another group (5). These comparisons show the ability of AutoSmarTrace to provide consistent persistence lengths (Fig.…”

“…This discrepancy likely arises from the heterogeneous structure of this network-forming collagen, compared with the fibril-forming types I and III collagens. It has been shown that type IV has an inhomogeneous flexibility profile: in particular, the persistence length is significantly lower (∼10 nm) towards the N-terminus of the protein (7, 28). Because of this, and avoidance of chain overlap in chain selection, it may be that more rigid portions of collagen IV chains are retained by the chain selection algorithm, while more flexible regions have a greater likelihood of exclusion from further analysis.…”

“…AFM tips with a 160 kHz resonance frequency and 5 N/m force constant (MikroMasch, HQ: NSC14/AL BS) were used. AFM images of the other collagen samples were similarly obtained: rat type I collagen images and collagen types I and III deposited from 20 mM acetic acid are from reference (6), while type IV collagen images are from reference (7).…”

“…The persistence length of chains varies between images from 20 nm to 200 nm at 5 nm intervals with an equal number of images per persistence length. This range encompasses experimentally measured persistence lengths for a variety of semiflexible polymers, including DNA, collagen and tropomyosin (4)(5)(6)(7)22). Chain width varied between images, with a value between three to six pixels.…”

“…The persistence length can be obtained by imaging individual filaments and determining the length scale over which thermal fluctuations tend to randomize the direction of the filament’s backbone. For semiflexible filaments such as DNA and molecular collagen, the persistence length is on the order of 10-100 nm (4-7), and thus atomic force microscopy (AFM) has become the technique of choice for conformational imaging, as it has sufficient spatial resolution to resolve the contour of the backbone of these filaments (1-2 nm in diameter). For thicker, more rigid filaments, fluorescence microscopy can be used to image fluctuations of filament backbones (8, 9).…”