No abstract

BackgroundTriple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required.ResultsHere, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils.ConclusionsUsing a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0228-7) contains supplementary material, which is available to authorized users.

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.

No abstract

No abstract