The formation of condensed matter typically involves a trade-off between structural order and flexibility. As the extent and directionality of interactions between atomic or molecular components increase, materials generally become more ordered but less compliant, and vice versa. Nevertheless, high levels of structural order and flexibility are not necessarily mutually exclusive; there are many biological (such as microtubules1,2, flagella3, viruses4,5) and synthetic assemblies (for example, dynamic molecular crystals6,7,8,9 and frameworks10,11,12,13) that can undergo considerable structural transformations without losing their crystalline order and that have remarkable mechanical properties8,14,15 that are useful in diverse applications, such as selective sorption16, separation17, sensing18 and mechanoactuation19. However, the extent of structural changes and the elasticity of such flexible crystals are constrained by the necessity to maintain a continuous network of bonding interactions between the constituents of the lattice. Consequently, even the most dynamic porous materials tend to be brittle and isolated as microcrystalline powders14, whereas flexible organic or inorganic molecular crystals cannot expand without fracturing. Owing to their rigidity, crystalline materials rarely display self-healing behaviour20. Here we report that macromolecular ferritin crystals with integrated hydrogel polymers can isotropically expand to 180 per cent of their original dimensions and more than 500 per cent of their original volume while retaining periodic order and faceted Wulff morphologies. Even after the separation of neighbouring ferritin molecules by 50 ångströms upon lattice expansion, specific molecular contacts between them can be reformed upon lattice contraction, resulting in the recovery of atomic-level periodicity and the highest-resolution ferritin structure reported so far. Dynamic bonding interactions between the hydrogel network and the ferritin molecules endow the crystals with the ability to resist fragmentation and self-heal efficiently, whereas the chemical tailorability of the ferritin molecules enables the creation of chemically and mechanically differentiated domains within single crystals.
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We thank the following colleagues for assistance: R. Alberstein for data processing; A. Rheingold, C. Moore and M. Gembicky for XRD; S. Weigand, T. Weiss and I. Rajkovic for SAXS; W.-J. Rappel for confocal microscopy; Z. Hu for performing the nanoindentation experiments. This work was primarily funded by the US Department of Energy, DOE (BES, Division of Materials Sciences, Biomolecular Materials Program, DE-SC0003844 to F.A.T.). Additional funding was provided by NSF (DMR-1602537 to F.A.T. for SAXS studies). Crystallographic data were collected at Stanford Synchrotron Radiation Lightsource (SSRL) and the Crystallography Facility of the University of California, San Diego. SAXS data were collected at SSRL and the Advanced Photon Source. SSRL and the Advanced Photon Source are supported by the DOE Office of Science, Office of Basic Energy Sciences under contracts DE-AC02-76SF00515 and DE-AC02-06CH11357, respectively.
L.Z., J.B.B. and F.A.T. have submitted a patent application based on the work described here.
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Extended data figures and tables
Extended Data Fig. 1 Distribution of electrostatic charge on the surface of ferritin and size distribution of ferritin in solution.
a, pH-dependent zeta potentials of ferritin, determined by dynamic-light-scattering measurements. b, Dynamic-light-scattering profile of ferritin (200 μM) in a solution of 50 mM HEPES (pH 7.0). c, Representation of the electrostatic charge distribution on the ferritin surface, as viewed along the two-, three- and four-fold symmetry axes. Positive (+5kBT/e) and negative (−5kBT/e) charges are shown in blue and red, respectively. kB, Boltzmann constant; e, electron charge.
Extended Data Fig. 2 Molecular diffusion and polymerization in ferritin crystals, monitored using confocal microscopy.
a, Diffusion of rhodamine B into a ferritin crystal over 15 min. b, c, In crystallo polymerization of the hydrogel network, monitored through the decrease of integrated pyranine fluorescence (green fluorescence channel). The corresponding bright-field (DIC) images show the diffusion of the aqueous NaCl solution into the crystal. The ring-shaped diffusion front becomes evident at time t = 108 s and disappears by t = 216 s. The crystal expands by approximately 5% (edge length) during polymerization. Scale bars in a and b correspond to 100 μm. d, Scanning electron microscopy images of native ferritin crystals (top) and crystal–hydrogel hybrids (bottom).
a, 19F-NMR spectrum, showing peak assignments for the trifluoroacetic acid standard, free 2-(trifluoromethyl)acrylic acid, and 2-(trifluoromethyl)acrylic acid incorporated into the polymer. b, Diagram illustrating the experimental protocol for the quantification of 2-(trifluoromethyl)acrylic acid uptake into the crystal lattice. The concentration of the 2-(trifluoromethyl)acrylic acid in the crystal lattice (155.6 mM) is approximately the same as its concentration in the soaking solution (see Methods for details).
a, b, Continuous expansion of two different crystal–hydrogel hybrids in deionized water, monitored using confocal microscopy. Crystal facets are still discernible after expansion for more than 2 h. Scale bars correspond to 100 μm. c, Ferritin release into the solution from expanding crystal–hydrogel hybrids (n > 10,000) over about 4 h. Negligible ferritin release is observed until about 1 h. Protein concentrations were determined using the Bradford assay. d, Confocal microscopy images of highly expanded crystal–hydrogel hybrids, showing the structural deterioration of the facets and the edges.
Extended Data Fig. 5 Expansion and contraction behaviour of crystal–hydrogel hybrids in the presence of different metal ions.
a, Light micrographs of the crystal–hydrogel hybrids at different stages of expansion and contraction in response to different metal ions. b, XRD patterns (T = 273 K) of expanded crystal–hydrogel hybrids, acquired upon contraction with different metal ions. Contraction with divalent cations (Ca, Mg, Cd, Zn, Ni and Co) reproducibly leads to the recovery of the full atomic-level order, whereas contraction with monovalent cations (Li, Na and K) only reinstates low-order diffraction peaks.
Extended Data Fig. 6 Successive expansion–contraction cycles for a single ferritin crystal–hydrogel hybrid.
Light micrographs of a hybrid crystal at pre- and post-expansion stages in each cycle are shown on the left, and the corresponding changes in edge length upon expansion–contraction are shown on the right. The separation between the major ticks of the ruler is 100 μm. The crystal expands to a lesser extent during the first expansion cycle, which we ascribe to residual CaCl2 (which forms strong polymer–polymer and protein–protein interactions) remaining in the solution that is transferred on the loop along with the crystal. The subsequent variability in the rate and extent of expansion is attributed to the different amounts of residual NaCl transferred in each cycle.
a, Alternative monomer combinations that yield successful in crystallo polymerization and crystal expansion. b, Monomer combinations that lead to crystal dissolution during polymerization. c, A crystal soaked in a solution containing polyacrylate (molecular weight, Mw = 2,100 Da) dissolves upon being transferred into water. The separation between the major ticks of the ruler is 100 μm. MBAm, N,N′-methylenebis(acrylamide).
a, Schematic diagram of the microfluidic chip. b, Side-view representations of the microfluidic chip. c, Photograph of the microfluidic chip, mounted on beamline 4-2 at SSRL. d, Single-crystal SAXS diffraction patterns observed at different stages of crystal expansion and contraction. The Miller indices of each visible spot are indicated. Reflections with the highest signal-to-noise ratio (I/σI) are circled in red. e, Spot profiles of the highest-I/σI reflections indicated in d.
a, Light-microscopy images showing the fragmentation of a native ferritin crystal and of a crystal–hydrogel hybrid upon application of external force with a needle at the location indicated with the arrow. The separation between the major ticks of the ruler is 100 μm. b, Temperature dependence of the SAXS profiles of native ferritin crystals and crystal–hydrogel hybrids. The small-angle reflections (that is, periodic order) in both samples are maintained at 80 °C (the maximal temperature experimentally attainable). c, Determination of the hardness and reduced modulus of native ferritin crystals and crystal–hydrogel hybrids using atomic force microscopy nanoindentation measurements. d, Light-microscopy images showing the expansion and contraction of a crystal–hydrogel hybrid containing Fe-loaded ferritin molecules. The separation between the major ticks of the ruler is 100 μm.
Video 1: In-crystallo formation of hydrogel network monitored by pyranine quenching (see Extended Data Fig. 2 for static snapshots)
To monitor the process of in-crystallo polymerization, a ferritin crystal infused with polymer precursors pyranine is imaged by confocal microscopy, using both fluorescent (green) and differential interference contrast (DIC) channels. 10 µL polymerization solution is added onto the crystal to initiate in-crystallo polymerization. Fluorescence from pyranine rapidly decreases in the first minute, indicating that the polymerization process was complete in <2 min. Polymerization is followed by the intrusion of aqueous NaCl solution into the lattice; the movement of the solvent front into the center of the crystal is observed as a ring that progressively becomes smaller.
Video 2: Structural evolution of a ferritin crystal-hydrogel hybrid during polymerization, expansion and contraction (see Main Text Fig. 2 for static snapshots)
A ferritin crystal infused with polymer precursors was placed on a microscopic ruler and imaged by light microscopy. 10 µL of the polymerization solution is added to initiate in-crystallo polymerization. The diffusion ring of the aqueous NaCl is clearly visible. After polymerization, the crystal is placed onto a clean ruler, deionized water is added and crystal-hydrogel hybrid expansion proceeds for ca. 10 min, whereby the crystal grows to ca. 140% of its original dimensions. Contraction is then mediated by NaCl addition, and a further incremental amount of contraction is achieved by CaCl2 addition.
Video 3: Examples of self-healing behavior in ferritin crystal-hydrogel hybrids (see Main Text Figs. 4a and 4b for static snapshots)
This video depicts three crystals (Crystals 1, 2 and 3) that undergo self-healing. Crystal 1 and Crystal 2 are crystal-hydrogel hybrids that develop cracks during contraction with CaCl2, and then undergo self-healing. In the case of Crystal 3, cracks appear during polymerization but are healed spontaneously. These cracks continually reappear and disappear during crystal expansion, and are aggravated for a brief period during the rapid, NaCl-induced contraction, but they eventually self-heal almost completely.
Video 4: Expansion and contraction behavior of functionalized ferritin crystals (see Extended Data Fig. 9d, and Main Text Figs. 4d and 4e for static snapshots)
Part 1: A crystal-hydrogel hybrid containing Fe-loaded ferritin expands in water and contracts upon addition of NaCl. Part 2: An expandable core/expandable shell crystal-hydrogel hybrid (core is labeled with rhodamine) expands in water and then contracts upon addition of NaCl. Part 3: An non-expandable core/expandable shell crystal-hydrogel hybrid (core is labeled with rhodamine and crosslinked with glutaraldehyde) undergoes shell-fragmentation upon transfer into water.
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Zhang, L., Bailey, J.B., Subramanian, R.H. et al. Hyperexpandable, self-healing macromolecular crystals with integrated polymer networks. Nature 557, 86–91 (2018). https://doi.org/10.1038/s41586-018-0057-7
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