Photo-responsive azobenzene interactions promote hierarchical self-assembly of collagen triple-helical peptides to various higher-order structures

Abstract

Collagen is an essential structural protein in animal tissues and plays key roles in cellular modulation. We investigated methods to discover collagen model peptides (CMPs) that would self-assemble into triple helices and then grow into supramolecular organizations with diverse morphological features, which would be valuable as biomaterials. This challenging undertaking was achieved by placing azobenzene groups on the ends of the CMPs, (GPO) _n (n = 3-10), Azo-(GPO) _n . In a dilute aqueous solution (80 μM), CD spectra indicated that the Azo-(GPO) _n (n > 4) formed triple helices due to strong hydrophobic azobenzene interactions, and that helix stability was increased with the peptide segment length. The resulting triple helices induced a specific azobenzene orientation through turned and twisted configurations as shown by CD spectra. TEM observations for the same solutions disclosed the morphologies for the Azo-CMPs. Azo-(GPO)₃, having the shortest peptide segment, showed no nanostructure, both Azo-(GPO)₄ and Azo-(GPO)₅ provided consistent well-developed nanofiber structures resembling the natural collagen fibers, and Azo-(GPO) _n s (n = 6-10) grew into flexible rod-like micelle fibers. In addition, alkyl chain-attached C _m Azo-(GPO)₅ displayed a toroidal morphology, and Azp-deg-(GPO)₅ having a hydrophilic spacer assembled into a bilayer vesicle structure. These diverse morphological features are considered to be due to the characteristics of the pre-organized triple helix units. Photo-isomerization of the azobenzene moiety brought about the disappearance of such characteristic nano-architectures. When the solution concentration was increased up to 1 wt%, only Azo-(GPO)₄ and Azo-(GPO)₅ spontaneously formed hydrogels exhibiting a satisfactory gel-to-sol transition upon UV irradiation.

Fig. 1. Chemical structures of the azobenzene-terminated collagen-mimetic oligopeptides: Azo-(GPO)_n (n = 3–10), Azo-deg-(GPO)₅, and C_mAzo-(GPO)₅ (m = 6 and 12).

Fig. 2. (a) Changes in the CD spectra of Azo-(GPO)₅ (trans-form) in water upon incubation at 4 °C after the thermal treatment at 60–90 °C, at which the peptides denature. (b and c) Denote time courses of the [θ]₂₂₅ and [θ]₃₀₇ values, respectively, for aqueous Azo-(GPO)_n (trans-form, n = 3–10) and Azo-deg-(GPO)₅ (trans-form) at 4 °C. [peptide] = 80 μM.

Fig. 3. TEM images stained by phosphotungstic acid for Azo-(GPO)_n (trans-form) (n = 3 (a), 4 (b), 5 (c), 6 (d), 7 (e), 8 (f), 9 (g), and 10 (h)), Azo-deg-(GPO)₅ (trans-form) (i), and C_mAzo-(GPO)₅ (trans-form) (m = 6 (j) and 12 (k)) incubated for 24 h at 4 °C.

Fig. 4. Proposed model for a hierarchical self-assembly system from azobenzene-terminated collagen-mimetic oligopeptides.

Fig. 5. (a) Changes in the absorption spectra of Azo-(GPO)₅ upon UV irradiation (365 nm) in water at 4 °C. [peptide] = 80 μM. (b) Photo-isomerization processes for Azo-(GPO)_n (n = 3–10) and Azo-deg-(GPO)₅ in water at 4 °C.

Fig. 6. (a) Changes in the CD spectra of Azo-(GPO)₅ upon UV irradiation (365 nm) in water at 4 °C. [peptide] = 80 μM. (b and c) Denote time courses of the [θ]₂₂₅ and [θ]₃₀₇ values, respectively, for Azo-(GPO)_n (n = 4–10) and Azo-deg-(GPO)₅ in water at 4 °C during UV irradiation. [peptide] = 80 μM.

Fig. 7. (a) Dynamic storage (G′) and loss (G′′) moduli for Azo-(GPO)₅-hydrogel (6 wt%) as a function of the strain (frequency: 6 rad s⁻¹) at 4 °C before (●, ▲) and after (○, △) UV irradiation. (b) G′ and G′′ moduli for Azo-(GPO)₅-hydrogel (6 wt%) as a function of the frequency (strain: 0.1%) at 4 °C. (c) Photographs of Azo-(GPO)₅-hydrogel before and after UV irradiation.