Abstract
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.
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The data supporting the findings of this study are available within the paper and its Supplementary Information files.
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Acknowledgements
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.
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D.J.P., C.J.K. and J.G.S. conceived of the project and mentored research activity, as well as contributed to the writing of the manuscript. D.W. carried out peptide synthesis, gold-nanoparticle synthesis and peptide conjugation with subsequent assembly into nanoparticle–bundle chains, and bundle and bundlemer polymer formation and characterization. D.W. also contributed to the writing of the manuscript. J.L. and N.S. contributed to characterization via TEM, cryoTEM, POM and SANS, with N.S. also contributing to the writing of the manuscript. B.P.S. and N.I.H. performed peptide synthesis, molecular characterization, rod formation and PEG conjugation. Y.T. performed AFM characterization. J.C. performed STORM characterization and contributed to the writing of the manuscript. H.V.Z. performed the computational design and modelling of peptides.
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Extended data figures and tables
Extended Data Fig. 1 Structures of peptides 1–7.
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.
Extended Data Fig. 2 The thiol–maleimide click reaction.
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.
Extended Data Fig. 3 SANS of peptide rods with different linker chemistries.
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.
Extended Data Fig. 4 Estimation of the persistence length of rigid-rod bundle chains.
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.
Extended Data Fig. 6 Chemical structures of bundlemer organic linkers.
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.
Extended Data Fig. 7 Hydrogel network rheology.
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.
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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). https://doi.org/10.1038/s41586-019-1683-4
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DOI: https://doi.org/10.1038/s41586-019-1683-4
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