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. 2006 Apr;7(4):1187-95.
doi: 10.1021/bm051002k.

Refolding hydrogels self-assembled from N-(2-hydroxypropyl)methacrylamide graft copolymers by antiparallel coiled-coil formation

Jiyuan Yang  1 , Chunyu Xu, Chun Wang, Jindrich Kopecek
Affiliations

Refolding hydrogels self-assembled from N-(2-hydroxypropyl)methacrylamide graft copolymers by antiparallel coiled-coil formation

Jiyuan Yang et al. Biomacromolecules. 2006 Apr.

Abstract

A novel hybrid hydrogel system based on N-(2-hydroxypropyl)methacrylamide copolymers was proposed. It consisted of the hydrophilic polymer backbone and a pair of oppositely charged peptide grafts. Two distinct pentaheptad peptides (CCE and CCK) were anticipated to create a dimerization motif and serve as physical cross-linkers. Consequently, the graft copolymers CCE-P and CCK-P self-assembled into hybrid hydrogels in situ; the process was modulated by the formation of antiparallel heterodimeric coiled-coils. This approach possesses an advantage to decrease the steric hindrance of the polymer backbone on the "in-register" alignment of peptide grafts. Indeed, equimolar mixtures of the graft copolymers, CCE-P/CCK-P, have been observed to self-assemble into hydrogels in PBS solution at neutral pH at concentrations as low as 0.1 wt %. Circular dichroism spectroscopy, sedimentation equilibrium experiments, and microrheology revealed that the self-assembly process corresponded to the two-stranded alpha-helical coiled-coil formation between CCE and CCK. Moreover, the formation of hybrid hydrogels was reversible. Denaturation of the coiled-coil domains with guanidine hydrochloride (GdnHCl) solutions resulted in disassembly of the hydrogels. Removal of GdnHCl by dialysis caused coiled-coil refolding and hydrogel reassembly. Scanning electron microscopy results demonstrated that the concentration of the graft copolymers had a significant impact on the structure and morphology of self-assembled hydrogels.

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Figures

Figure 1
Figure 1
Helical wheel representation of two-stranded, antiparallel α-helical coiled coils formed by the dimerization of CCE and CCK. The view is shown looking down the superhelical axis from the N-terminus of CCE and from the C-terminus of the CCK. CC denotes the coiled-coil peptides. E and K denote peptides in which most of the e and g positions are occupied by either glutamic acid or lysine, respectively. The sequences are written in the one-letter amino acid code. Positions a and d of the heptad repeat are underlined and form the hydrophobic core of the coiled-coil.
Figure 2
Figure 2
(A) Synthesis of HPMA graft copolymers and (B) schematic representation of hydrogel formation through antiparallel heterodimeric coiled-coil association.
Figure 3
Figure 3
Analytical reverse-phase HPLC chromatograms of CCK peptide, HPMA copolymer precursor (P), and their conjugate, CCK-P. The chromatogram was developed using ZOBRAX C18 column (250 × 4.6 mm) with Agilent 1100 system at a flow rate of 1 ml/min using a gradient 2–60% acetonitrile over 30 min.
Figure 4
Figure 4
Circular dichroism (CD) spectra of CCK, CCE, and their equimolar mixture CCE/CCK with (A) and without (B) 50% TFE of the α-helix-inducing solvent, trifluoroethanol (TFE). (C) Effect of mole ratio in the mixture on the ellipticity at 222 nm (no TFE). The spectra were recorded at 25°C in 50 mM Na2HPO4/NaH2PO4, 100 mM KCl, pH 7.0. All peptide concentrations were approximately 0.45 mg/ml (100 μM).
Figure 5
Figure 5
Characterization and thermal stability of HPMA graft copolymers. (A) CD spectra of CCE-P, CCK-P, and their equimolar mixture CCE-P/CCK-P. The spectra were recorded in PBS (50 mM Na2HPO4/NaH2PO4, 100 mM KCl, pH 7) with peptide concentration 100 μM. (B) Temperature effect on the secondary structure of CCE/CCK and CCE-P/CCK-P in PBS as monitored by change of molar ellipticity at 222 nm.
Figure 6
Figure 6
The pH dependence of secondary structure of (A) CCE; (B) CCK; (C) CCE/CCK (1:1); and (D) Content of α-helix as a function of pH.
Figure 7
Figure 7
Thermal stability of CCE and CCK at different pH.
Figure 8
Figure 8
Mean-squared displacements (MSD, <Δr2(t)>) of particles involved in CCE-P/CCK-P systems as a function of time (1% w/v).
Figure 9
Figure 9
(A) CD spectra of equimolar mixture CCK-P/CCE-P before (full diamonds) and after (empty triangles) denaturation by guanidine hydrochloride at 25°C with 20 μM peptide concentration, and after removing of GdnHCl via dialysis (empty squares). (B) Dependence of molar ellipticity at 222 nm of CCE-P/CCK-P on concentration of GdnHCl.
Figure 10
Figure 10
SEM images of hybrid hydrogels self-assembled from equimolar solutions of CCE-P and CCK-P in PBS (A) 0.5 wt. % HPMA copolymer solution; (B) one component (0.5 wt. % CCE-P) only; (C) 0.1 wt.% CCE-P/CCK-P; (D) 0.5 wt.% CCE-P/CCK-P; (E) 1.0 wt.% CCE-P/CCK-P; (F) 5.0 wt.% CCE-P/CCK-P.
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