The engineering of biological molecules is a key concept in the design of highly functional, sophisticated soft materials. Biomolecules exhibit a wide range of functions and structures, including chemical recognition (of enzyme substrates or adhesive ligands1, for instance), exquisite nanostructures (composed of peptides2, proteins3 or nucleic acids4), and unusual mechanical properties (such as silk-like strength3, stiffness5, viscoelasticity6 and resiliency7). Here we combine the computational design of physical (noncovalent) interactions with pathway-dependent, hierarchical ‘click’ covalent assembly to produce hybrid synthetic peptide-based polymers. The nanometre-scale monomeric units of these polymers are homotetrameric, α-helical bundles of low-molecular-weight peptides. These bundled monomers, or ‘bundlemers’, can be designed to provide complete control of the stability, size and spatial display of chemical functionalities. The protein-like structure of the bundle allows precise positioning of covalent linkages between the ends of distinct bundlemers, resulting in polymers with interesting and controllable physical characteristics, such as rigid rods, semiflexible or kinked chains, and thermally responsive hydrogel networks. Chain stiffness can be controlled by varying only the linkage. Furthermore, by controlling the amino acid sequence along the bundlemer periphery, we use specific amino acid side chains, including non-natural ‘click’ chemistry functionalities, to conjugate moieties into a desired pattern, enabling the creation of a wide variety of hybrid nanomaterials.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data supporting the findings of this study are available within the paper and its Supplementary Information files.
Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 23, 47–55 (2005).
Egelman, E. H. et al. Structural plasticity of helical nanotubes based on coiled-coil assemblies. Structure 23, 280–289 (2015).
Yang, Y. J., Holmberg, A. L. & Olsen, B. D. Artificially engineered protein polymers. Annu. Rev. Chem. Biomol. Eng. 8, 549–575 (2017).
Seeman, N. C. & Sleiman, H. F. DNA nanotechnology. Nat. Rev. Mater. 3, 17068 (2018).
Ding, Z. Z. et al. Simulation of ECM with silk and chitosan nanocomposite materials. J. Mater. Chem. B 5, 4789–4796 (2017).
Dooling, L. J., Buck, M. E., Zhang, W.-B. & Tirrell, D. A. Programming molecular association and viscoelastic behavior in protein networks. Adv. Mater. 28, 4651–4657 (2016).
Li, L. Q., Tong, Z. X., Jia, X. Q. & Kiick, K. L. Resilin-like polypeptide hydrogels engineered for versatile biological function. Soft Matter 9, 665–673 (2013).
Bryson, J. W. et al. Protein design—a hierarchical approach. Science 270, 935–941 (1995).
Huang, P.-S. et al. High thermodynamic stability of parametrically designed helical bundles. Science 346, 481–485 (2014).
Zhang, H. V. et al. Computationally designed peptides for self-assembly of nanostructured lattices. Sci. Adv. 2, e1600307 (2016).
Dogic, Z. & Fraden, S. Ordered phases of filamentous viruses. Curr. Opin. Colloid Interface Sci. 11, 47–55 (2006).
Dogic, Z. Surface freezing and a two-step pathway of the isotropic-smectic phase transition in colloidal rods. Phys. Rev. Lett. 91, 165701 (2003).
Gittes, F., Mickey, B., Nettleton, J. & Howard, J. Flexural rigidity of microtubules and actin-filaments measured from thermal fluctuations in shape. J. Cell Biol. 120, 923–934 (1993).
Zhang, R., Kumar, N., Ross, J. L., Gardel, M. L. & de Pablo, J. J. Interplay of structure, elasticity, and dynamics in actin-based nematic materials. Proc. Natl Acad. Sci. USA 115, E124–E133 (2018).
Zhao, J. G. et al. Freeze-thaw cycling induced isotropic-nematic coexistence of amyloid fibrils suspensions. Langmuir 32, 2492–2499 (2016).
Bharti, B., Fameau, A. L., Rubinstein, M. & Velev, O. D. Nanocapillarity-mediated magnetic assembly of nanoparticles into ultraflexible filaments and reconfigurable networks. Nat. Mater. 14, 1104–1109 (2015).
Hume, J. et al. Engineered coiled-coil protein microfibers. Biomacromolecules 15, 3503–3510 (2014).
Papapostolou, D. et al. Engineering nanoscale order into a designed protein fiber. Proc. Natl Acad. Sci. USA 104, 10853–10858 (2007).
Dong, H., Paramonov, S. E. & Hartgerink, J. D. Self-assembly of alpha-helical coiled coil nanofibers. J. Am. Chem. Soc. 130, 13691–13695 (2008).
Usov, I., Adamcik, J. & Mezzenga, R. Polymorphism complexity and handedness inversion in serum albumin amyloid fibrils. ACS Nano 7, 10465–10474 (2013).
Usov, I. & Mezzenga, R. FiberApp: an open-source software for tracking and analyzing polymers, filaments, biomacromolecules, and fibrous objects. Macromolecules 48, 1269–1280 (2015).
VandenAkker, C. C., Engel, M. F. M., Velikov, K. P., Bonn, M. & Koenderink, G. H. Morphology and persistence length of amyloid fibrils are correlated to peptide molecular structure. J. Am. Chem. Soc. 133, 18030–18033 (2011).
Aggeli, A. et al. Hierarchical self-assembly of chiral rod-like molecules as a model for peptide β-sheet tapes, ribbons, fibrils, and fibers. Proc. Natl Acad. Sci. USA 98, 11857–11862 (2001).
Manning, G. S. The persistence length of DNA is reached from the persistence length of its null isomer through an internal electrostatic stretching force. Biophys. J. 91, 3607–3616 (2006).
Averick, S. et al. Cooperative, reversible self-assembly of covalently pre-linked proteins into giant fibrous structures. Angew. Chem. Int. Ed. 53, 8050–8055 (2014).
Oshaben, K. M. & Horne, W. S. Tuning assembly size in peptide-based supramolecular polymers by modulation of subunit association affinity. Biomacromolecules 15, 1436–1442 (2014).
Thomas, F., Burgess, N. C., Thomson, A. R. & Woolfson, D. N. Controlling the assembly of coiled-coil peptide nanotubes. Angew. Chem. Int. Ed. 55, 987–991 (2016).
Ok, J. M. et al. Control of periodic defect arrays of 8CB (4′-n-octyl-4-cyano-biphenyl) liquid crystals by multi-directional rubbing. Soft Matter 9, 10135–10140 (2013).
Dogic, Z. Filamentous phages as a model system in soft matter physics. Front. Microbiol. 7, 1013 (2016).
Huang, B., Bates, M. & Zhuang, X. Super-resolution fluorescence microscopy. Annu. Rev. Biochem. 78, 993–1016 (2009).
Kline, S. R. Reduction and analysis of SANS and USANS data using IGOR Pro. J. Appl. Cryst. 39, 895–900 (2006).
Guinier, A. & Fournet, G. Small-Angle Scattering of X-Rays (Wiley, 1955).
Pedersen, J. S. & Schurtenberger, P. Scattering functions of semiflexible polymers with and without excluded volume effects. Macromolecules 29, 7602–7612 (1996).
Chen, W., Butler, P. D. & Magid, L. J. Incorporating intermicellar interactions in the fitting of SANS data from cationic wormlike micelles. Langmuir 22, 6539–6548 (2006).
Jung, J. & Mezzenga, R. Liquid crystalline phase behavior of protein fibers in water: experiments versus theory. Langmuir 26, 504–514 (2010).
Haider, M. J., Zhang, H., Sinha, N., Saven, J. G. & Pochan, D. J. Self-assembly and soluble aggregate behavior of computationally designed coiled-coil peptide bundles. Soft Matter 14, 5488–5496 (2018).
Käs, J., Strey, H. & Sackmann, E. Direct imaging of reptation for semiflexible actin filaments. Nature 368, 226–229 (1994).
Boal, D. H. Mechanics of the Cell (Cambridge Univ. Press, 2012).
Falvo, M. R. et al. Manipulation of individual viruses: friction and mechanical properties. Biophys. J. 72, 1396–1403 (1997).
Primary funding was provided by the Department of Energy, Office of Basic Energy Sciences, Biomolecular Materials Program under grants DE-SC0019355 and DE-SC0019282. D.J.P. and N.S. acknowledge stipend support and support for neutron-scattering experiments under cooperative agreements 70NANB12H239 and 70NANB17H302 from the National Institute of Standards and Technology (NIST), US Department of Commerce. D.J.P. and N.S. also acknowledge the support of the NIST in providing neutron research facilities. Neutron facilities are supported in part by the National Science Foundation (NSF) under agreement DMR-0944772. We acknowledge the support of the National Institutes of Health (NIH), grant RO1 EB006006, and the University of Delaware NIH Centers of Biomedical Research Excellence (COBRE) grants 1P30.GM110758 and 1P20.RR017716 for instrument resources. We acknowledge the Delaware IDeA Network of Biomedical Research Excellence grant P20 GM103446 for support of the Delaware Biotechnology Institute. J.G.S. acknowledges partial support from NSF CHE 1709518 and the Penn Laboratory for Research on the Structure of Matter (MRSEC; grant NSF DMR-1120901). D.J.P. and J.L. acknowledge stipend support from the NSF under grant DMREF-1629156. D.J.P., J.G.S., Y.T. and H.Z. acknowledge stipend support from the NSF under grants DMR-1234161 and DMR-1235084. The statements, findings, conclusions and recommendations herein are those of the authors and do not necessarily reflect the view of NIST, the US Department of Commerce, the US Department of Energy, or the University of Delaware. D.W., D.J.P. and C.J.K. acknowledge internal research support from the University of Delaware.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Vincent Conticello, Shuguang Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
The single-letter amino acid sequences of peptides 1–7 are shown at the top, noting the peptides in which maleimide (Mal) has been chemically added to the N terminus, and those in which lysine amino acids (K) have been modified with the structures shown at the bottom. The unmodified amino acid sequences of peptide 1, peptide 2 (without an N-terminal cysteine) and peptide 6 have previously been denoted P622_6 (ref. 10), BNDL1 (ref. 10) and BNDL2 (ref. 37), respectively.
R1 and R2 represent the possible remainders of the molecular structures. The thiol–maleimide reaction is catalysed by either a base or a nucleophile (Nu) catalyst.
a, Scattering from rigid rods (blue squares) and the corresponding rigid cylinder fit (black curve). Rigid rods (identical to those in Fig. 1b) were assembled in 25 mM pH 6 phosphate buffer prepared in deuterated water at 20 °C. b, Scattering from semi-flexible fibres (purple triangles) and the corresponding flexible cylinder fit (black curve). Semi-flexible chains (identical to those shown in Fig. 1e) were dissolved in 25 mM pH 6 phosphate buffer prepared in deuterated water at 20 °C. In each case, R is the fitted cylinder radius of the corresponding model.
The estimation was carried out using FibreApp tracking and the analysis software of ref. 21. a, CryoTEM of rigid-rod bundle chains. b, The same rigid-rod bundle chains after software tracking. The red trace is the last rod tracked with the software in the image and is used, along with the earlier blue traces, in the stiffness analyses. c, Plot of calculated mean-squared end-to-end distance (MSED) between contour segments along the tracked rod (blue squares), and the corresponding MSED fit (black curve) between contour segments. This fit is based on a worm-like chain model in two dimensions with the following theoretical dependence: ⟨R2⟩ = 4λ[l – 2λ(1 – e–l/2λ)], where λ is the persistence length and R is the direct distance between any pair of segments along a contour separated by arc length l. The persistence length is estimated to be 33.4 ± 0.4 μm. d, An alternative method for estimating the persistence length of very stiff, one-dimensional objects is the mean-squared midpoint displacement (MSMD). This plot shows the calculated MSMD between contour segments along the tracked rod (blue squares) and the corresponding MSMD fit (black curve). This fit is based on an equation that describes the behaviour of a midpoint deviation along a rod: ⟨ux2⟩ = l3/48λ, where ⟨ux2⟩ is the MSMD between any pair of segments along a tracked-rod contour, separated by an arc length l, assuming that the displacements are small in comparison with the corresponding arc lengths (|ux| is much less than l). This method estimates the persistence length of the rod to be 41.3 ± 0.5 μm.
Extended Data Fig. 5 Plot showing persistence length versus mass per unit length for various 1D polymers and molecular assemblies.
The plot is adapted with permission from ref. 16, Springer Nature Limited. For peptide-bundlemer rigid rods, the persistence length was estimated from our cryoTEM data using the methods of ref. 21. Other persistence lengths were taken from the following references: hydrocarbon polymers, ref. 16; fd and M13 viruses, refs. 11,12; DNA, refs. 16,24; actin, refs. 13,38; tobacco mosaic virus, refs. 16,39,40; microtubulin, refs. 13,39,40; thin (diameter 2–3 nm) and thick (diameter 4–6 nm) twisted bovine serum albumin (BSA) fibres, ref. 20; β-lactoglobulin β-sheet-rich twisted fibrils (with diameters ranging from 1 nm to 6 nm, with a mean of roughly 3 nm), ref. 11,22; amino acid β-sheet ribbons with low stacking (producing ribbon diameters of roughly 4 nm) and high stacking (producing ribbons with diameters of 4–8 nm), ref. 23.
a, Structure of PETMP for the formation of semiflexible or kinked chains. b, Chemical structure of four-arm PEG tetrathiol (20 kDa) for hydrogel formation.
Hydrogels were composed of peptides 1 or 5 (Extended Data Fig. 1) linked with four-arm PEG tetrathiol (Extended Data Fig. 6). We monitored the storage modulus G′ (filled symbols) and loss modulus G″ (open symbols) as functions of temperature (left) and as a result of the cycling of temperature (right); in the latter case, temperature ramps were performed over 5 min between isothermal measurements of 2-min duration. a, Peptide 1, with Tm = 55 °C. Peptide 1 produces a temperature-reversible hydrogel owing to the low melting temperature of the designed peptide. b, Peptide 5, with Tm = 85 °C. The hydrogel produced with peptide 5 is stable to approximately 85 °C and shows much more rigid gel properties at all temperatures tested.
About this article
Cite this article
Wu, D., Sinha, N., Lee, J. et al. Polymers with controlled assembly and rigidity made with click-functional peptide bundles. Nature 574, 658–662 (2019) doi:10.1038/s41586-019-1683-4