Abstract
Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.
Highlights
Fibrillar collagens are the dominant proteins in mammalian tissue extracellular matrix
Whilst the distribution of proline ring conformations found from X-ray diffraction (XRD) structural analysis of model collagen peptides can be expected to broadly reflect the conformational propensity of proline rings, limits in resolution mean that XRD structural models cannot be expected to reproduce accurate ring conformation populations
In the collagen model peptides of this study, we expect Cγ chemical shifts approximately in this range, the precise value being determined by the endo:exo ratio for the imino residue ring being observed in the NMR spectrum, i.e. we will measure chemical shift values of nendo·δendo+ nexo·δexo where nendo and nexo are the fractions of rings in the endo and exo conformational states respectively, and δendo and δexo are the isotropic 13C shifts of the pure endo and exo conformations
Summary
Fibrillar collagens are the dominant proteins in mammalian tissue extracellular matrix. Synthetic peptides containing sufficient (GPO)n and (GPP)n can self-assemble in water to give a collagen-like triple helix. These structures have helix parameters close to 7/2 helical symmetry (20.0 Å axial repeat, synonymous with 75)[6,7,8,9], while fibre X-ray diffraction of native tendon collagen has indicated a looser 10/3 helical symmetry (28.6 Å axial repeat, synonymous with 107) in collagen proteins in vivo[10]. The conformation of prolyl rings in (GPO)n and (GPP)n is a well-studied feature of triple helical peptides, with the expectation that general principles in these model systems will be replicated in native collagen triple helices. Experiments[11,12] and calculations[13] show clearly that the exo conformation of prolyl rings in the Yaa position stabilises the collagen triple helix, the fact Yaa prolines in native collagenous tissues are predominantly modified to exo-preferring hydroxyproline (exo-preferring through a combination of a gauche effect and an n → π* interaction)[14,15,16]
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