Naghmeh Rezaei

Using optical tweezers to study mechanical properties of collagen

The mechanical response of biological molecules at the microscopic level contributes significantly to their function. Optical tweezers are instruments that enable scientists to study mechanical properties at microscopic levels. They are based on a highly focused laser beam that creates a trap for microscopic objects such as dielectric spheres, viruses, bacteria, living cells and organelles, and then manipulates them by applying forces in the picoNewton range (a range that is biologically relevant). In this work, mechanical properties of single collagen molecules are studied using optical tweezers. We discuss the challenges of stretching single collagen proteins, whose length is much less than the size of the microspheres used as manipulation handles, and show how instrumental design and biochemistry can be used to overcome these challenges.

Development and characterization of a eukaryotic expression system for human type II procollagen

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.

Probing multiscale mechanics of collagen with optical tweezers

How the molecular structure of the structural, extracellular matrix protein collagen correlates with its mechanical properties at different hierarchical structural levels is not known. We demonstrate the utility of optical tweezers to probe collagen’s mechanical response throughout its assembly hierarchy, from single molecule force-extension measurements through microrheology measurements on solutions of collagen molecules, collagen fibrillar gels and gelatin. These experiments enable the determination of collagen’s flexibility, mechanics, and timescales and strengths of interaction at different levels of hierarchy, information critical to developing models of how collagen’s physiological function and stability are influenced by its chemical composition. By investigating how the viscoelastic properties of collagen are affected by the presence of telopeptides, protein domains that strongly influence fibril formation, we demonstrate that these play a role in conferring transient elasticity to collagen solutions.

Conformational flexibility of collagen molecules: composition and environmental effects

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 proteins mechanical properties. Our results show that types I, II and III collagens - the key fibrillar varieties - exhibit 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 collagens 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 collagens 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.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-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 varieties – exhibit 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. 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.

Environmentally Controlled Curvature of Single Collagen Proteins

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 proteins 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 collagens 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 collagens 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.

Additional file 1: Figure S1. of Development and characterization of a eukaryotic expression system for human type II procollagen

Results of mass spectrometric analysis on the recombinant human type II procollagen, demonstrating protein sequence coverage of COL2A1 and expected posttranslational modifications. (PDF 70 kb)