Self-assembly of synthetic collagen triple helices

Abstract

Collagen is the most abundant protein in animals and the major component of connective tissues. Although collagen isolated from natural sources has long served as the basis for some biomaterials, natural collagen is difficult to modify and can engender pathogenic and immunological side effects. Collagen comprises a helix of three strands. Triple helices derived from synthetic peptides are much shorter (<10 nm) than natural collagen (approximately 300 nm), limiting their utility. Here, we describe the synthesis of short collagen fragments in which the three strands are held in a staggered array by disulfide bonds. Data from CD spectroscopy, dynamic light scattering, analytical ultracentrifugation, atomic force microscopy, and transmission electron microscopy indicate that these "sticky-ended" fragments self-assemble via intermolecular triple-helix formation. The resulting fibrils resemble natural collagen, and some are longer (>400 nm) than any known collagen. We anticipate that our self-assembly strategy can provide synthetic collagen-mimetic materials for a variety of applications.

Fig. 1.

Structure and self-assembly of fragments 1 and 2. (A) Amino acid sequence of fragments 1 and 2. The glycine residue preceding the adjacent cysteines in the α2 strand establishes the requisite register for triple-helix formation with the identical α1 and α1′ strands. Hyp, (2S,4R)-4-hydroxyproline. (B) Representation of the self-assembly process; green, red, and blue are used simply to distinguish individual fragments.

Fig. 2.

Scheme for the synthesis of self-assembling fragments 1 and 2. tBu, tert-butyl; Acm, acetamidomethyl; Bu, butyl; TFE, 2,2,2-trifluoroethanol; iPr, isopropyl; HMPA, 4-hydroxymethylphenoxyacetyl; PEGA, polyethylene glycol acrylamide copolymer.

Fig. 3.

CD data for assemblies of fragments 1 and 2. (A) Spectra of assembled 1 and 2 (200 μM) at 4°C. (Inset) 1, 70 μM; 2, 100 μM. (B) Thermal denaturation of assembled 1 and 2 (200 μM), monitored at 226 nm. (C) Rate of assembly of fragments 1 and 2 (30 and 150 μM), over 90 min, monitored at 226 nm after a rapid transition from 90°C to 10°C. The [Θ]_max for each sample was calculated as the difference between the ellipticity before denaturation (100% assembled) and the first data point at 10°C, thus omitting the initial jump in ellipticity (which was not resolvable) (47). The value of percent assembled was then calculated as the ratio of the observed recovery to [Θ]_max. Data in A–C were from solutions in 50 mM HOAc(aq) at pH 2.9. (D) Thermal denaturation of assembled 1 and 2 (200 μM) in 67:33 MeOH/68 mM HOAc(aq) monitored at 226 nm.

Fig. 4.

AFM and TEM images for assemblies of fragments 1 and 2. (A) AFM height image of (1)_n. (B) AFM height image of isolated (1)_n (Upper) and section analysis of the data along the direction indicated by the arrow (Lower). (C) TEM image of (1)_n after rotary shadowing with platinum. (Inset) An isolated fibril. (D) The same as A, except for (2)_n. (E) The same as B, except for (2)_n. (F) The same as C, except for (2)_n.