Assembly Of Collagen Triple Helices Research Articles

Effects of glycosylated (2S,4R)-hydroxyproline on the stability and assembly of collagen triple helices.

Functionalized collagen-mimetic peptides (CMPs) have been widely used in the preparation of collagen-related biomaterials. Among the reported results, the induced noncovalent interactions between the implanted functional groups or moieties were frequently the key elements to promote the self-assembly of small CMPs. In this work, we designed and synthesized a series of glycosylated CMPs in which 4-O-[β-D-galactopyranosyl]-(2S,4R)-4-hydroxyproline (Hyp(Gal)) was incorporated to explore the effects of glycosylation on the stability and assembly of collagen triple helices. Circular dichroism measurements showed that glycosylation of hydroxyproline slightly destabilized the collagen triple helices, but did not reduce their refolding rate. Compared to non-glycosylated CMPs, the incorporation of Hyp(Gal) speeded up the assembly of CMPs, indicating that this modification could assist the self-assembly of CMPs into higher-order structures, such as fibrils. O-Galactosylation of hydroxyproline imposes contrary effects on the triple helix stability and the self-assembly rate of collagen triple helices, exhibiting a piece of important and useful information for designing collagen-related biomaterials. Our finding also suggests that instead of stabilizing the triple helical conformation of CMPs, installing additional forces between CMPs could be a crucial factor to promote the assembly of CMPs into large-scale constructs.

A FRET-Based Method for Measurement of Yeast Septin Filament Formation In Vitro

A FRET-Based Method for Measurement of Yeast Septin Filament Formation In Vitro

Investigating the Mechanism of Collagen Self-Assembly with Microrheology

Collagen is the predominant protein in vertebrates, where it comprises more than 1/4 of the total protein and performs structural and mechanical tasks in the extracellular matrix and connective tissues. Formed intracellularly as a triple helical protein, collagen undergoes self-assembly extracellularly into fibrils, which in turn contribute to higher-order structures. Self-assembly of collagen into well-ordered fibrils can be replicated with appropriate solvent conditions in vitro, implying that the keys to self-association are found within its sequence.We perform microrheology experiments on collagen systems, using optically trapped particles as probes of local, microscale viscoelasticity. Our initial experiments investigated the time-dependent development of viscoelastic heterogeneity in collagen systems as they undergo self-assembly from proteins in solution into fibrillar structures.1 Here, we examine alterations in collagen's chemical composition that influence its self-assembly. We find that the removal of collagen's nonhelical “telopeptide” ends significantly reduces elasticity of collagen solutions at timescales from ∼10 msec to ∼1 sec. While in the acidic solutions of these experiments collagen does not assemble into fibrils, our results nonetheless provide insight into the catalytic mechanism of these short domains on fibril formation. The removal of telopeptides has long been known to slow down fibril formation, and telopeptides have previously been postulated to transiently associate with other chains in solution,2 thus providing “docking points” from which lateral assembly of collagen triple helices into fibrils can proceed. Here, our microrheology experiments provide direct evidence of increased strength and duration of interprotein contact arising from the presence of telopeptides, critical in catalyzing self-assembly of fibrillar collagen systems.1 M. Shayegan and N.R. Forde, PLoS ONE8, e70590 (2013)2 N. Kuznetsova and S. Leikin, J. Biol. Chem.274, 36083 (1999); D.J. Prockop and A. Fertala, J. Biol. Chem.273, 15598 (1998).

Liquid crystalline phases of sonicated type I collagen.

The assembly properties of concentrated solutions of type I collagen molecules are compared before and after a 5-min sonication, breaking the 300-nm triple helices into short segments of about 20 nm, with a strong polydispersity. The collagen concentration of these solutions, sonicated or not, was increased up to 100 mg/ml by slow evaporation of the solvent. Whereas the non-sonicated solutions remain isotropic, the sonicated solutions transform after a few hours into a twisted liquid crystalline phase, well recognizable in polarizing microscopy. The evidence of a twisted assembly of collagen triple helices in vitro is new and relevant in a biological context since it was reported in various collagen matrices.

Proposed mechanism for the biological assembly of collagen triple helix.

If the three polypeptide chains of collagen had terminal segments with structures which facilitated rapid association and were then discarded, a puzzle might be solved. Such a mechanism could explain why biosynthesis of collagen takes minutes in vivo but much longer in vitro.