Abstract
Purpose
To explore the relationship between the structure of block polypeptides and their self-assembly into hydrogels. To investigate structural parameters that influence hydrogel formation and physical properties.
Methods
Three ABA triblock and two AB diblock coiled-coil containing polypeptides were designed and biologically synthesized. The triblock polypeptides had two terminal coiled-coil (A) domains and a central random coil (B) segment. The coiled-coil domains were different in their lengths, and tyrosine residues were incorporated at selected solvent-exposed positions in order to increase the overall hydrophobicity of the coiled-coil domains. The secondary structures of these polypeptides were characterized by circular dichroism and analytical ultracentrifugation. The formation of hydrogel structures was evaluated by microrheology and scanning electron microscopy.
Results
Hydrogels self-assembled from the triblock polypeptides, and had interconnected network microstructures. Hydrogel formation was reversible. Denaturation of coiled-coil domains by guanidine hydrochloride (GdnHCl) resulted in disassembly of the hydrogels. Removal of GdnHCl by dialysis caused coiled-coil refolding and hydrogel reassembly.
Conclusions
Protein ABA triblock polypeptides composed of a central random block flanked by two coiled-coil forming sequences self-assembled into hydrogels. Hydrogel formation and physical properties may be manipulated by choosing the structure and changing the length of the coiled-coil blocks. These self-assembling systems have a potential as in-situ forming depots for protein delivery.
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References
G. Tae, J. A. Kornfield, and J. A. Hubbell. Sustained release of human growth hormone from in situ forming hydrogels using self-assembly of fluoroalkyl-ended poly(ethylene glycol). Biomaterials 26:5259–5266 (2005).
E. Ruel-Gariépy and J. C. Leroux. In situ-forming hydrogels—review of temperature-sensitive systems. Eur. J. Pharm. Biopharm. 58:409–426 (2004).
E. Behravesh, K. Zygourakis, and A. G. Mikos. Adhesion and migration of marrow-derived osteoblasts on injectable in situ crosslinkable poly(propylene fumarate-co-ethylene glycol)-based hydrogels with a covalently linked RGDS peptide. J. Biomed. Mater. Res. A 65:260–270 (2003).
B. Balakrishnan and A. Jayakrishnan. Self-cross-linking biopolymers as injectable in situ forming biodegradable scaffolds. Biomaterials 26:3941–3951 (2005).
A. Motulsky, M. Lafleur, A. C. Couffin-Hoarau, D. Hoarau, F. Boury, J. P. Benoit, and J. C. Leroux. Characterization and biocompatibility of organogels based on L-alanine for parenteral drug delivery implants. Biomaterials 26:6242–6253 (2005).
L. Zhang, E. M. Furst, and K. L. Kiick. Manipulation of hydrogel assembly and growth factor delivery via the use of peptide–polysaccharide interactions. J. Control. Release 114:130–142 (2006).
C. Wang, R. J. Stewart, and J. Kopeček. Hybrid hydrogels self-assembled from synthetic polymers and coiled-coil domains. Nature 397:417–420 (1999).
J. Kopeček, A. Tang, C. Wang, and R. J. Stewart. De novo design of biomedical polymers: Hybrids from synthetic macromolecules and genetically engineered protein domains. Macromol. Symp. 174:31–42 (2001).
C. Wang, J. Kopeček, and R. J. Stewart. Hybrid hydrogels crosslinked by genetically engineered coiled-coil block proteins. Biomacromolecules 2:912–920 (2001).
J. Yang, C. Xu, P. Kopeèková, and J. Kopeček. Hybrid hydrogels self-assembled from HPMA copolymers containing peptide grafts. Macromol. Biosci. 6:201–209 (2006).
J. Yang, C. Xu, C. Wang, and J. Kopeček. Refolding hydrogels self-assembled from N-(2-hydroxypropyl)methacrylamide graft copolymers by antiparallel coiled-coil formation. Biomacromolecules 7:1187–1195 (2006).
C. Xu and J. Kopeček. Self-assembling hydrogels. Polym. Bull. 58:53–63 (2007).
Y. B. Yu. Coiled-coils: stability, specificity, and drug delivery potential. Adv. Drug Deliv. Rev. 54:1113–1129 (2002).
W. A. Petka, J. L. Harden, K. P. McGrath, D. Wirtz, and D. A. Tirrell. Reversible hydrogels from self-assembling artificial proteins. Science 281:389–392 (1998).
C. Xu, V. Breedveld, and J. Kopeček. Reversible hydrogels from self-assembling genetically engineered protein block copolymers. Biomacromolecules 6:1739–1749 (2005).
L. Serrano, M. Bycroft, and A. R. Fersht. Aromatic–aromatic interactions and protein stability. Investigation by double-mutant cycles. J. Mol. Biol. 218:465–475 (1991).
F. Schafer, D. Deluca, U. Majdic, J. Kirchner, M. Schliwa, L. Moroder, and G. Woehlke. A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core. EMBO J. 22:450–458 (2003).
S. Adio, J. Reth, F. Bathe, and G. Woehlke. Review: regulation mechanisms of kinesin-1. J. Muscle Res. Cell Motil. 27:153–160 (2006).
S. R. Campion, R. K. Matsunami, D. A. Engler, and S. K. Niyogi. Biochemical properties of site-directed mutants of human epidermal growth factor: importance of solvent-exposed hydrophobic residues of the amino-terminal domain in receptor binding. Biochemistry 29:9988–9993 (1990).
M. Walther, R. Wiesner, and H. Kuhn. Investigations into calcium-dependent membrane association of 15-lipoxygenase-1. Mechanistic roles of surface-exposed hydrophobic amino acids and calcium. J. Biol. Chem. 279:3717–3725 (2004).
E. Hochuli. Purification of recombinant proteins with metal chelate adsorbent. Genet. Eng. (N Y) 12:87–98 (1990).
C. R. Cantor and P. R. Schimmel. Biophysical Chemistry, W. H. Freeman and Company, New York, 1980.
D. A. Yphantis. Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3:297–317 (1964).
M. L. Johnson, J. J. Correia, D. A. Ypahantis, and H. R. Halvorson. Analysis of data from the analytical ultracentrifuge by non-linear least squares techniques. Biophys. J. 36:575–588 (1981).
T. M. Laue, B. D. Shah, T. M. Ridgeway, and S. L. Pelletier. Computer aided interpretation of analytical sedimentation data for proteins. In S. E. Harding, A. J. Rowe, and J. C. Horton (eds.), Ultracentrifugation in Biochemistry and Polymer Science, The Royal Society of Chemistry, Cambridge, 1992, pp. 90–125.
J. C. Crocker, M. T. Valentine, E. R. Weeks, T. Gisler, P. D. Kaplan, A. G. Yodh, and D. A. Weitz. Two-point microrheology of inhomogeneous soft materials. Phys. Rev. Lett. 85:888–891 (2000).
P. B. Harbury, T. Zhang, P. S. Kim, and T. Alber. A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 262:1401–1407 (1993).
J. Y. Su, R. S. Hodges, and C. M. Kay. Effect of chain length on the formation and stability of synthetic alpha-helical coiled coils. Biochemistry 33:15501–15510 (1994).
J. C. Crocker and D. G. Grier. Methods of digital video microscopy for colloidal studies. J. Colloid Interface Sci. 179:298–310 (1996).
T. G. Mason and D. A. Weitz. Optical measurements of frequency-dependent linear viscoelastic moduli of complex fluids. Phys. Rev. Lett. 74:1250–1253 (1995).
F. C. MacKintosh and C. F. Schmidt. Microrheology. Curr. Opin. Colloid Interface Sci. 4:300–307 (1999).
W. Shen, K. Zhang, J. A. Kornfield, and D. A. Tirrell. Tuning the erosion rate of artificial protein hydrogels through control of network topology. Nat. Mater. 5:153–158 (2006).
K. Wagschal, B. Tripet, and R. S. Hodges. De novo design of a model peptide sequence to examine the effects of single amino acid substitutions in the hydrophobic core on both stability and oligomerization state of coiled-coils. J. Mol. Biol. 285:785–803 (1999).
K. Wagschal, B. Tripet, P. Lavigne, C. Mant, and R. S. Hodges. The role of position a in determining the stability and oligomerization state of alpha-helical coiled-coils: 20 amino acid stability coefficients in the hydrophobic core of proteins. Protein Sci. 8:2312–2329 (1999).
B. Tripet, K. Wagschal, P. Lavigne, C. Mant, and R. S. Hodges. Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position “d”. J. Mol. Biol. 300:377–402 (2000).
A. E. Keating, V. N. Malashkevich, B. Tidor, and P. S. Kim. Side-chain repacking calculations for predicting structures and stabilities of heterodimeric coiled coils. Proc. Natl. Acad. Sci. U. S. A. 98:14825–14830 (2001).
J. Kopeček. Swell gels. Nature 417:388–391 (2002).
Acknowledgements
We thank Drs. Bruce Yu and Jiyuan Yang for valuable discussions, Dr. David Tirrell for the kind gift of plasmid pUC18-RC, and Jon Callahan for critical reading of the manuscript. The research was supported in part by NIH grant EB005288.
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Xu, C., Kopeček, J. Genetically Engineered Block Copolymers: Influence of the Length and Structure of the Coiled-Coil Blocks on Hydrogel Self-Assembly. Pharm Res 25, 674–682 (2008). https://doi.org/10.1007/s11095-007-9343-z
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DOI: https://doi.org/10.1007/s11095-007-9343-z