Sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures

Abstract

The process of self-assembly of the triple-helical peptide (Pro-Hyp-Gly)(10) into higher order structure resembles the nucleation-growth mechanism of collagen fibril formation in many features, but the irregular morphology of the self-assembled peptide contrasts with the ordered fibers and networks formed by collagen in vivo. The amino acid sequence in the central region of the (Pro-Hyp-Gly)(10) peptide was varied and found to affect the kinetics of self-assembly and nature of the higher order structure formed. Single amino acid changes in the central triplet produced irregular higher order structures similar to (Pro-Hyp-Gly)(10), but the rate of self-association was markedly delayed by a single change in one Pro to Ala or Leu. The introduction of a Hyp-rich hydrophobic sequence from type IV collagen resulted in a more regular suprastructure of extended fibers that sometimes showed supercoiling and branching features similar to those seen for type IV collagen in the basement membrane network. Several peptides, where central Pro-Hyp sequences were replaced by charged residues or a nine-residue hydrophobic region from type III collagen, lost the ability to self-associate under standard conditions. The inability to self-assemble likely results from loss of imino acids, and lack of an appropriate distribution of hydrophobic/electrostatic residues. The effect of replacement of a single Gly residue was also examined, as a model for collagen diseases such as osteogenesis imperfecta and Alport syndrome. Unexpectedly, the Gly to Ala replacement interfered with self-assembly of (Pro-Hyp-Gly)(10), while the peptide with a Gly to Ser substitution self-associated to form a fibrillar structure.

Figure 1.

(A) The temperature dependence of the self-assembly process for peptide O14A (7 mg/mL, PBS, pH = 7) monitored by turbidity measurements (absorbance at 313 nm). (B) Turbidity curves showing temperature induced self-assembly of collagen peptides (7 mg/mL, PBS, pH = 7): (1) (POG)₁₀ at 58°C, (2) O14A at 52°C, (3) P13L at 51°C (dashed line), and (4) P13A at 51°C. The inset shows turbidity (absorbance at 313 nm) measured as a function of time at 51°C for the peptide P13L at a higher concentration of ∼16 mg/mL. (C) DSC profile of the thermal unfolding of (POG)₁₀ (○), O14A (■), and P13L (▲).

Figure 2.

Electron micrographs of the self-assembled structures formed by the collagen peptides: (A) (Pro-Hyp-Gly)₁₀, (B) O14A, and (C) P13L. Each peptide (7 mg/mL, PBS, pH 7) was incubated at its optimum temperature for aggregation. The sample was collected after the onset of the plateau phase for microscopy.

Figure 3.

(A) Turbidity curves showing self-assembly (7 mg/mL, PBS, pH = 7) of a set of triple-helical peptides with a type IV sequence, differing only in their terminal residues: (1) T4α5–491Y at T = 40°C, (2) T4α5–491Y′ at T = 44°C, (3) T4α5–491 at T = 40°C. Inset is the turbidity curve for the peptide T4α5–491, showing the slower kinetics of self-assembly with a timescale of hours. (B) Turbidity curves showing the temperature dependence of self-assembly for peptide T4α5–491Y (7 mg/mL, PBS, pH = 7). (C) DSC profiles of thermal unfolding of peptide T4α5–491Y′ showing two distinct transitions.

Figure 4.

Electron micrograph of the self-assembled structures formed by the set of collagen peptides with a type IV sequence. (A) T4α5–491, (B) T4α5–491Y, and (C) T4α5–491Y′. A magnified view of a portion of C is shown in D, with small arrows indicating the branching and supercoiled nature of the structure.

Figure 5.

Electron micrograph of the self-assembled structure formed by the peptide (POG)₁₀ with one Gly replaced by Ser. The peptide (7 mg/mL, PBS, pH = 7) undergoes aggregation resulting in a slightly turbid solution when incubated at 25°C for 1 d.