Anisotropic dynamics and mechanics of macromolecular crystals containing lattice-patterned polymer networks

Containing Lattice-Patterned Polymer Networks

Article

Anisotropic Dynamics and Mechanics of Macromolecular Crystals

Kenneth Han, Jake B. Bailey, Ling Zhang, and F. Akif Tezcan*

Cite This: https://dx.doi.org/10.1021/jacs.0c10065

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sı Supporting Information

ABSTRACT: The mechanical and functional properties of many

crystalline materials depend on cooperative changes in lattice

arrangements in response to external perturbations. However, the

ﬂexibility and adaptiveness of crystalline materials are limited.

Additionally, the bottom-up, molecular-level design of crystals with

desired dynamic and mechanical properties at the macroscopic

level remains a considerable challenge. To address these

challenges, we had previously integrated mesoporous, cubic ferritin

crystals with hydrogel networks, resulting in hybrid materials

(

polymer-integrated crystals or PIX) which could undergo

dramatic structural changes while maintaining crystalline perio-

dicity and display eﬃcient self-healing. The dynamics and

mechanics of these ferritin-PIX were devoid of directionality, which is an important attribute of many molecular and macroscopic

materials/devices. In this study, we report that such directionality can be achieved through the use of ferritin crystals with anisotropic

symmetries (rhombohedral or trigonal), which enable the templated formation of patterned hydrogel networks in crystallo. The

resulting PIX expand and contract anisotropically without losing crystallinity, undergo prompt bending motions in response to

stimuli, and self-heal eﬃciently, capturing some of the essential features of sophisticated biological devices like skeletal muscles.

INTRODUCTION

A major goal in materials science is to apply chemical design at

the atomic/molecular scale to generate collective structural,

dynamic and responsive properties of molecular crystals are

■

typically obtained a posteriori (rather than by de novo design)

and on a case-by-case basis. Consequently, the molecular

building blocks or the self-assembly conditions cannot be easily

altered to obtain diﬀerent structural and mechanical properties

at the macroscopic scale.

dynamic, and mechanical properties at the macroscopic scale.

The corresponding transfer and ampliﬁcation of atomic/

molecular-level information require the molecular building

blocks to be organized and appropriately interconnected over

Soft polymeric materials like hydrogels provide a complete

contrast to crystalline materials in many aspects. They are

ﬂexible, responsive, and their mechanical properties can be

readily modulated by chemical design or physical manipu-

−4

multiple length scales.

Crystalline materials provide a

distinct advantage in this regard in that they are composed

of only one or few components arranged periodically,

possessing both short and long-range order to allow structural

and mechanical coupling in a cooperative manner. Indeed,

there is a growing number of dynamic molecular crystals,

ﬂexible protein lattices, and porous framework materials,

28,29

lation.

However, these attributes are attained precisely

because polymeric materials are devoid of the structural order

and coherence of crystalline materials, leading ultimately to a

lack of mechanical strength and cooperativity. Previously, we

surmised that the complementary but mutually exclusive

advantages of crystalline and polymeric materials (crystals:

structural order/strength/cooperativity; hydrogels: ﬂexibility/

responsiveness/self-healing) could be combined if molecular

crystals and hydrogel networks were chemically and structur-

9,10

which can promptly undergo dramatic lattice transformations

and motions in response to external stimuli, with promising

1−13

applications in actuation,

gas sorption and separa-

4−16

17,18

19,20

tion,

sensing,

and controlled release,

among

,9,10

others.

of exceptions,

However, with relatively few but growing number

1−25

these dynamic crystalline materials tend to

be brittle, cannot undergo large changes in volume at the

macroscale without mechanical failure, or self-heal. These

limitations are due to the fact that the lattice components need

to be continuously interconnected during structural trans-

formations to maintain crystallinity. While there have been

advances in exploiting reticular chemistry approaches to

Received: September 20, 2020

26,27

deliberately design ﬂexible porous networks,

the precise

XXXX American Chemical Society

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

chain ferritin (Figure 1 a).

Article

ally integrated. As a proof-of-principle platform, we used the

face-centered-cubic (fcc, F432 symmetry, a ≈ 180 Å) crystals

of the quasi-spherical, 24meric, iron-storage protein human H-

chemical responsiveness and eﬃcient self-healing behavior.

The anisotropic ferritin-PIX thus provide a compelling

example for the molecular-scale design of hierarchical materials

with bespoke macroscale properties.

31,32

The cubic lattice is formed

RESULTS AND DISCUSSION

■

Preparation, Characterization, and Self-Assembly of

Ferritin Modiﬁed with RAFT Agents. To generate

anisotropic ferritin-PIX, we set out to prepare a ferritin variant

that was site-selectively modiﬁed with RAFT (reversible

6,37

addition−fragmentation chain-transfer) agents.

Our ra-

tionale was based on our original expectation that the RAFT-

modiﬁed ferritins could enable the controlled growth of

polymer networks in spatially well-deﬁned locations within the

36,37

protein lattice. RAFT polymerization

provides excellent

compatibility with aqueous solutions and acrylate monomers,

does not require transition metal ions (which may interfere

with ferritin self-assembly), and has been commonly used to

generate covalent protein−polymer hybrids with high

8,39

eﬃciency via graft-from strategies.

Accordingly, we

synthesized a Cys-speciﬁc maleimide-functionalized trithiocar-

bonate RAFT agent (Figure 1b and S1). We used this agent to

Figure 1. Ferritin as a building block for polymer-integrated crystals

PIX). (a) Schematic representation for the generation and isotropic

expansion/contraction properties of cubic ferritin PIX homoge-

C157

site-selectively label the ferritin variant,

ferritin, which bears

(

a single set of surface-exposed Cys residues (24 total, at

positions 157) ﬂanking the ferritin C symmetry axes (Figure

neously infused with a polyacrylate network. (b) Schematic for the

site-speciﬁc conjugation of a maleimide-functionalized RAFT agent to

1b and S2). The graft-from growth of the pA polymer from the

C157

RAFT

ferritin. The resulting conjugate,

ferritin, can assemble into

modiﬁed variant (termed

ferritin) could be induced by the

isotropic (cubic) or anisotropic (rhombohedral) crystals in a pH-

dependent manner (scale bars: 100 μm).

radical initiators VA-044 or APS/TEMED and was conﬁrmed

by SDS-PAGE electrophoresis and gel permeation chromatog-

raphy (GPC) (Figures S3 and S4).

We next examined the self-assembly of

RAFT

through the Ca -D84/Q86-mediated association of the C

ferritin into 3D

symmetric interfaces of each ferritin molecule with 12

crystals. Under typical conditions used for Ca -mediated

ferritin crystallization (≥5 mM CaCl , pH 8.0), we obtained

octahedron-shaped, fcc crystals (F432, a = 179.9 Å, PDB ID:

neighbors. The ferritin lattice, like many protein crystals, is

mesoporous, with continuously linked, nm-sized channels that

account for an interstitial solvent content of 39%. This porosity

allows the full permeation of ferritin crystals with acrylate

polymer precursors and the subsequent formation of a

pervasive polyacrylate (pA) hydrogel network within the

lattice (Figure 1a). Owing to the extensive noncovalent

interactions between pA side chains and the surfaces of ferritin

molecules, the resulting materials (termed Polymer-Integrated

Crystals or PIX) behave essentially as singular chemical units

that exhibit unprecedented material properties. For example,

pA-ferritin PIX can reversibly expand and contract in response

to changes in ionic strength by nearly 600% in volume without

losing crystalline order (Figure 1a) and display eﬃcient self-

healing. However, as a result of the uniform distribution and

isotropic expansion/contraction of the pA network within the

protein lattice, the structural dynamics of the ﬁrst-generation

ferritin-PIX were also isotropic, meaning that they lacked

RAFT

6WYF) of

unmodiﬁed

ferritin that were isomorphous with those of

ferritin (Figures 1b and 2a). RAFT agents

C157

attached to the C157 side chains extend into the 6 nm wide,

cube-shaped cavities in the lattice and can be discerned in the

1.25-Å resolution crystal structure up to the amide group

(Figure 2a). When the solution pH is lowered to ≤6.5,

RAFT

ferritin molecules self-assemble into large (≥60 μm)

rhombohedron-shaped crystals (H32, a = b = 127.0 Å, c =

281.7 Å, PDB ID: 6WYG) which lack the 3D isotropy of the

fcc crystals (Figures 1b and 2b).

The rhombohedral ^R^A^F^Tferritin crystal lattice can be

considered as a layered structure (Figure 2b). The hexagonal

layers in the ab-plane are mediated by Ca -D84/Q86

interactions between each ferritin molecule and six neighbors

(Figure 2c), as in the cubic crystals. In contrast, the interlayer

interactions along the c-axis are formed by contacts between

the hydrophobic patches consisting of groups of four C157-

directionality. Inspired by the remarkable mechanics of

skeletal muscles, there has been extensive interest in designing

anisotropic soft-material platforms that display directional

RAFT moieties surrounding the ferritin C axes (Figure 2d).

These interactions further connect each ferritin molecule with

six additional neighbors in the c-direction, yielding a quasi-

hexagonal close-packed arrangement with a denser packing

(interstitial solvent content = 32.5%) than the cubic crystals.

34,35

motion and anisotropic mechanical properties.

Accord-

ingly, we asked whether it is possible to control the spatial

distribution of hydrogel networks within ferritin-PIX to achieve

directional/anisotropic dynamic behavior. Here, we report that

the hydrogel networks within PIX can indeed be patterned by

the orientation and structural details of the distinct protein−

protein interfaces in noncubic ferritin lattices. The resulting,

anisotropic ferritin-PIX with lattice-patterned hydrogel net-

works display directional expansion/contraction and rapid

bending motions, while retaining crystalline order, as well as

Electrostatic calculations show that at pH = 8, C surfaces of

ferritin are highly negatively charged and thus self-repulsive,

accounting for the fcc arrangement (Figure S5). Upon lowering

the pH to ≤6.5, the negative charge is mostly mitigated,

promoting hydrophobic interlayer interactions (Figure S5).

RAFT

Thus, although each

ferritin molecule is inherently

isotropic, the energetic balance/competition between diﬀerent

Journal of the American Chemical Society

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Article

Figure 2. Formation and structural properties of ^R^A^F^Tferritin crystals/PIX. Surface positions involved in Ca -mediated ferritin-ferritin interactions

at the ferritin C axes are highlighted in orange and the RAFT-labeled C157 patches at the ferritin C axes are shown in magenta. (a) The fcc

RAFT

(

F432) packing arrangement of the isotropic

ferritin crystals, along with a view of the lattice along the (200) plane and a close-up view of the

RAFT agents (magenta) attached to C157 positions. The 2F −F electron density map for a single conformation of the RAFT-labeled C157 site is

RAFT

contoured at 0.7σ. (b) The hexagonal-layered (H32) packing arrangement of the anisotropic

green hexagon. (c) Intralayer interactions between ferritin molecules in the (0001) plane oriented along the ab-plane are mediated by Ca ions

orange spheres) and two pairs of D84 and Q86 side chains. (d) Interlayer interactions, oriented along the c axis, are mediated by ferritin surfaces

that include hydrophobic patches (purple) formed by the RAFT agents. (e) Interlayer separation increases by ca. 3 Å after acrylate infusion. (f)

ferritin crystals. The (0001) plane is shown as a

(

RAFT

Formation of the pA within rhombohedral

ferritin crystals is monitored by confocal ﬂuorescence microscopy (left) through the disappearance

of pyranine ﬂuorescence, which is complete within 10 min (right, scale bars: 100 μm).

interactions (metal-mediated and hydrophobic) governing its

self-assembly yield both isotropic and anisotropic lattice

arrangements in a condition-dependent manner, similar to

dimensions increased by only 2 Å (unit cell: a = b = 128.9 Å, c

= 291.8 Å, PDB ID: 6WYH). This behavior is quite similar to

that of layered double hydroxide materials, which undergo

anisotropic lattice expansion/contraction upon exchange of the

40,41

what has been observed for spherical nanoparticles.

Anisotropic Dynamics of Rhombohedral ^R^A^F^TFerritin

PIX. There have been extensive eﬀorts toward designing

hydrogel-based materials that display muscle-like, directional

motion, and complex deformations in response to external

intercalating anions in the interlayer spaces. The 2.2-Å

RAFT

resolution structure of the acrylate-soaked

ferritin revealed

a striking picture in which the neighboring hexagonal ferritin

layers (i.e., the ab-planes) were separated from one another by

3−4 Å (Figure 2e). This expansion eliminates any observable

direct contact between the ferritin molecules along the c

direction (and increases the interstitial solvent content of the

4,35

stimuli.

However, hydrogels inherently undergo isotropic

volumetric changes. Therefore, multistep physical alignment/

patterning strategies and external ﬁelds have to be applied to

introduce anisotropic arrangements of polymer chains or

embedded particles to obtain directional behavior with

hydrogels.

of the rhombohedral

lattice from 32% to 37%), while the Ca -mediated intralayer

interactions remain intact. These ﬁndings highlight the ﬂuidity

of the interlayer interactions and the anisotropy inherent in the

rhombohedral crystals.

4,35,42−47

In our system, the anisotropic structure

RAFT

ferritin lattices and the speciﬁc

positioning of the RAFT agents in these lattices create a

unique opportunity to generate an anisotropic hydrogel

network solely via (one-step) molecular self-assembly and

potentially generate directional actuation.

The formation of the pA hydrogel network within ^R^A^F^Tferri-

tin crystals was eﬃciently mediated by radical initiators VA-

044 (0.2% w/v) or APS/TEMED (1% w/v). In-crystallo

polymerization was monitored by confocal microscopy,

whereby we followed the quenching of the ﬂuorescence of

pyranine molecules (λ_m_a_x= 512 nm) infused into the crystals

(Figure 2f, S6, and Video S1). The process was typically

To investigate this possibility, rhombohedral ^R^A^F^Tferritin

crystals were ﬁrst perfused with 1 M of acrylate monomers,

which caused no visible loss in the integrity of the crystals.

Interestingly, single-crystal X-ray diﬀraction (sc-XRD) meas-

urements indicated that this treatment caused a 10-Å

expansion of the lattice along the c axis whereas the a/b

complete in <2 min for a typical, 100 μm-sized crystal, but we

RAFT

incubated the acrylate-permeated

ferritin crystals with

radical initiators for at least 5 min to ensure full hydrogel

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

expansion was observed, conﬁrming that the pA polymer

Article

formation within the crystals. These experiments were carried

out in the presence of 4 M NaCl to prevent crystal expansion

matrix was responsible for the reversible expansion of PIX

(Figure S7). Although microcracks were sometimes visible

during pA-induced expansion/contraction, the faceted crystal

morphology was preserved throughout the process. Upon

during polymerization. As previously shown, the inclusion of

chemical cross-linkers like N,N′-methylenebis(acrylamide) was

unnecessary for the formation of a stable hydrogel owing to the

extensive interactions between the ferritin surface and the

carboxylate functional groups of pA, which yield a tightly

interwoven physical network.

addition of NaCl and/or CaCl

contracted and regained their original dimensions within 5 s

Figure 3a,b). The expansion/contraction process was fully

, the rhombohedral PIX

(

reversible over at least eight cycles as long as the expansion was

stopped before 2 min (Figure S8).

In a typical expansion experiment, the rhombohedral

RAFT

Importantly, the structural dynamics of rhombohedral

ferritin PIX were transferred into deionized water and

RAFT

ferritin PIX were highly anisotropic, as evidenced by (a)

monitored by light microscopy for 1−20 min (Figure 3a). The

expansion proceeded rapidly upon transfer and followed

the increase in the macroscopic aspect ratio of the crystals

long axis length

short axis length

biphasic kinetics (τ_f_a_s_t≤ 10 s; τ

> 50 s), with the PIX

(deﬁned as

) by over 50% after 1 min of expansion

slow

growing to nearly 200% of their original dimensions within 1

min (Figure 3b). When the same experiments were repeated

with propionate, a nonpolymerizable acrylate analog, no

and (b) concomitant changes in the facet angles from ∼56°

and ∼126° to ∼43° and ∼137°, respectively (Figure 3a and

Video S2). In contrast to rhombohedral PIX, the expansion

RAFT

and contraction of cubic

ferritin PIX were isotropic at all

times (Figure S9), suggesting that directional dynamics do not

stem simply from RAFT-polymerization per se but likely from

the higher density of the pA network in the interlayer

interfaces containing the RAFT agents within the rhombohe-

dral ferritin crystals. Indeed, upon assignment of crystal facet

indices, we found that the long crystal axis, which showed

disproportionate elongation compared to the short axis,

aligned with the c-axis of the lattice along which the interlayer

interfaces were oriented (Figure 3c).

To elucidate lattice dynamics in molecular detail, we carried

out time-dependent, small-angle X-ray scattering (SAXS)

RAFT

measurements on

ferritin PIX. In these experiments, in-

crystallo polymerization was initiated by X-ray irradiation, and

the diﬀraction patterns of >100 PIX suspended in sample

capillaries were collected. The SAXS symmetry was retained

(

Figure 3d,e). As in the case of light microscopy measure-

ments, the continuous increase in anisotropy during crystal

expansion was clearly evident in symmetry was retained

(

Figure 3d,e). As in the case of light microscopy measure-

ments, the continuous increase in anisotropy during crystal

expansion was clearly evident in the diﬀraction patterns. The

unit cell became a = b = 134.5 Å, c = 383.5 Å after 1 min of

expansion, corresponding to an increase in the microscopic

c axis length

a axis length

aspect ratio (deﬁned as

) by 25% and the cell volume

by 43% (Figure 3e,f). After 5 min expansion, the long-range

ferritin periodicity was still apparent from the presence of

strong (003) and (101) peaks, which yield a unit cell of a = b =

47.4 Å, c = 436.9 Å (Figure 3d,e). These values correspond to

Figure 3. Anisotropic expansion and contraction behavior of

increases in the cell aspect ratio and volume by 31% and 96%,

respectively, compared to unexpanded crystals (Figure 3f,g).

Consistent with light microscopy measurements, the kinetics

for the growth of unit cell dimensions and the increase in

longitudinal anisotropy was also nonmonotonic (Figure 3e,f).

We attribute this behavior to a fast initial expansion of the

dense pA network throughout the PIX, which attenuates as the

overall polymer density decreases and polymer chain mobility

RAFT

rhombohedral

expansion and contraction of a single rhombohedral

ferritin PIX. (a) Monitoring of the anisotropic

RAFT

ferritin PIX

by light microscopy. The separation between the major ticks of the

ruler is 100 μm. (b) The corresponding changes in long-axis length of

RAFT

the same

ferritin PIX during polymerization, expansion, and

contraction. (c) Facet indices and lattice orientation in rhombohedral

RAFT

ferritin PIX. (d) SAXS images collected at diﬀerent time points

RAFT

during the expansion of rhombohedral

ferritin PIX and (e) the

corresponding 1D SAXS proﬁles. The progression of peaks to lower

angles (due to expansion of the unit cell) is indicated with black

dashed lines. Peaks corresponding to the original lattice (due to

unexpanded crystals) are visible throughout the process and

designated with blue asterisks. (f) Changes in the unit cell dimensions

of rhombohedral PIX during expansion, calculated from the SAXS

proﬁles shown in part e. (g) Changes in the aspect ratio (i.e., the

anisotropy) of the unit cell of rhombohedral PIX during expansion.

Error bars: standard deviation of triplicate measurements.

increases. Time-dependent SAXS measurements were repeated

RAFT

for cubic

ferritin PIX, which conﬁrmed that the cubic

symmetry−thus the 3D isotropy−was retained throughout

expansion (Figure S10).

Taken together, our observations are consistent with an

anisotropic distribution of the pA polymer matrix within

RAFT

rhombohedral

ferritin crystals, which we (originally)

attributed to a combination of two factors: (1) the speciﬁc

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

generating a lamellar pattern (Figure 4 a). Consequently, the

Article

interlayer positions of the RAFT agents which promote

localized polymer growth and (2) the wide, weakly bound

interlayer interfaces, which are further enlarged upon soaking

with acrylate monomers. Both factors would lead to the

interlayer zones developing a denser matrix of pA polymer

compared to the tighter interfaces along the ab-planes, thus

hydration of the PIX produces a larger extent of lattice

expansion parallel to the c-axis compared to that in the ab-

plane (Figure 4a). Although the pA network displays varying

densities, it is continuous throughout the mesoporous lattice

and forms extensive interactions with the ferritin molecules.

The resulting dense mold thus allows the expanded ferritin

lattice to fully revert to its original dimensions upon NaCl/

CaCl -induced dehydration. In fact, the sc-XRD measurements

RAFT

show that

ferritin PIX fully regains near-atomic-level

crystallinity upon contraction after 5 min of expansion (Figure

b), meaning that ferritin molecules can return to their original

lattice positions and orientations after having separated from

one another by >20 Å in the ab-plane and >40 Å along the c-

axis.

Structural Basis of Anisotropic Polymer Distribution

in Ferritin PIX. We next examined if the anisotropic pA

RAFT

distribution in

ferritin PIX indeed could be ascribed to

localized polymer growth originating from the RAFT agents on

RAFT

ferritin surfaces. To this end, the

dissolved by treatment with ethylenediaminetetraacetic acid

EDTA) and analyzed by SDS-PAGE and GPC. Interestingly,

ferritin PIX were

(

RAFT

these

ferritin PIX samples showed no evidence of covalent

attachment between pA chains and ferritin molecules when in-

crystallo polymerization was induced with APS/TEMED and

only minimal yields of graft-from polymerization when VA-044

was used as a radical initiator (Figure S11). The drastically

diminished graft-from polymerization eﬃciencies are likely due

to the steric occlusion of the RAFT agents within the interlayer

interfaces and slower molecular diﬀusion within the crystals.

These observations implied that the inherent anisotropy of the

rhombohedral crystals was alone responsible for templating an

anisotropic hydrogel network in ferritin PIX.

_F_i_g_u_r_e₄_._A_n_i_s_o_t_r_o_p_i_c_e_x_p_a_n_s_i_o_n_/_c_o_n_t_r_a_c_t_i_o_n_o_f_r_h_o_m_b_o_h_e_d_r_a_lRAFT-

ferritin PIX is enabled by the anisotropic polymer matrix and is fully

reversible. (a) Schematic summary for the generation of the

anisotropic/layered pA network within rhombohedral ^R^A^F^Tferritin

crystals, which dictates the anisotropic structural dynamics. (b) sc-

RAFT

XRD image (at T = 298 K) of native rhombohedral

ferritin crystal

(

left), compared to those of a PIX contracted after 1 min of expansion

middle), and after 5 min of expansion (right). The diﬀraction limits

are indicated with red circles. Light micrographs of the crystals are

shown in the insets; the scale bar and separation between the major

ticks of the ruler are 100 μm.

An appropriate control system to test this possibility would

be ferritin crystals that are also rhombohedral but lack

Figure 5. Structural properties and anisotropy of ^Δ^Cferritin crystals/PIX with P3 21 symmetry. (a) ^Δ^CFerritin molecules along the ab-plane are

devoid of any intralayer interactions. The closest side chains, Q86 and D84, are 6.1 Å apart which precludes any salt-bridge interactions. (b)

Interlayer interactions, oriented along the c axis, are mediated by surfaces that include hydrophobic patches. The closest noncovalent interaction is

within 3.0 Å and formed by side chains K119 and E116. Where the H32 lattice hydrophobic patches would be are highlighted in green. (c) Light

micrographs of P3 21 symmetric PIX during expansion in water. The separation between the major ticks of the ruler is 100 μm. (d) Changes in the

crystal facet aspect ratios of P3 21 and H32 symmetric PIX during expansion display their respective anisotropic behavior. Error bars: standard

ΔC

deviation of triplicate measurements. (e) Facet indices and lattice orientation in trigonal ferritin PIX.

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Article

covalently attached RAFT agents. Since the RAFT agents are

directly involved in lattice packing interactions, we were not

able to obtain isomorphous rhombohedral crystals using

C157

unmodiﬁed

ferritin. Yet, in the course of exhaustive

screening, we found that a ferritin variant lacking Cys157

termed ΔC) formed lattices with trigonal symmetry (P3 21; a

(

b = 131.8 Å, c = 301.8 Å, PDB ID: 7K26) and a

rhombohedron-shaped crystal habit that is nearly identical to

RAFT

that of rhombohedral (i.e., H32-symmetric)

ferritin

crystals. The 2.7-Å resolution structure of the trigonal crystals

indeed revealed a similar hexagonal-layered packing arrange-

ment with an interstitial solvent content of 44.5% but also

indicated that the protein interfaces in these lattices

substantially diﬀer from those in their rhombohedral counter-

part. Notably, the lattice packing interactions between ferritin

molecules are mediated entirely by the interlayer interfaces

directed along the c axis, whereas the intralayer interfaces in

the ab-plane are ca. 6 Å wide at their narrowest point and

devoid of direct ferritin-ferritin contacts (Figure 5a,b). Thus, in

terms of the orientation of interstitial voids that can be ﬁlled

with the pA matrix, the trigonal and rhombohedral lattices are

orthogonal to one another.

Figure 6. Anisotropic mechanical and self-healing behavior of

RAFT

rhombohedral

ferritin PIX. (a) Light micrographs showing the

RAFT

bending motion of an expanded

ferritin PIX ﬂexing in response to

Ca ﬂux (oriented along the orange arrow). The separation between

the major ticks of the ruler in all images is 100 μm. (b) Schematic

Despite the relative mechanical fragility of the trigonal

crystals, we were able to ﬁnd conditions to form pA matrices

within them (Supporting Information). The expansion/

contraction properties of the resulting PIX were examined by

light microscopy, which revealed that they also displayed

description for the cation-induced bending motion of the rhombohe-

RAFT

dral

ferritin PIX due to the underlying hexagonal-layered lattice

arrangement and the anisotropic distribution of the polymer network.

RAFT

ferritin PIX are overwhelmingly

oriented in the direction of the ab-planes (orthogonal to the long

RAFT

anisotropic dynamics, but the direction of crystal expansion

crystal axis) and often spontaneously healed. (d)

ferritin PIX can

RAFT

was orthogonal to that observed with rhombohedral

ferri-

undergo susbtantial lamellar fracturing and accordion-like ﬂexing

tin PIX (Figure 5c−e and Video S3). Whereas the

rhombohedral PIX elongated to assume a lozenge shape

motions in response to Ca ﬂux.

(

with the acute facet angles decreasing from ∼60° to ∼45°,

orders of magnitude higher than those of recently reported

supramolecular and hydrogel systems with some of the fastest

Figure 3a) upon expansion, the trigonal PIX expanded toward

a more square-like shape (with the acute facet angles increasing

from ∼60° to 76°, Figure 5c), with the overall aspect ratios of

the two systems moving in opposite directions (Figure 5d).

These ﬁndings establish that 1) the anisotropy of the crystal

lattices and the underlying orientation/structure of the

protein−protein interfaces alone are suﬃcient for the

formation of anisotropic hydrogel networks within PIX, and

−

−1

reported actuation rates (1.5° s

and 0.14° min ,

49,50

respectively).

The rapid actuation by the PIX can be

ascribed to the high packing density and the structural

cooperativity of the integrated crystal-pA matrix.

Under certain circumstances like excessive bending or fast

RAFT

expansion/contraction, the rhombohedral

ferritin PIX

were observed to develop large fractures, sometimes >75 μm

in length and >10 μm in width (Figure 6c and Video S5).

Consistent with the mechanical anisotropy of these materials,

the defects were overwhelmingly oriented along the short

crystal axis (i.e., parallel to the hexagonal ferritin layers in the

ab-planes) (Figure 6c). Owing to the mobility of the hydrogel

matrix and its reversible interactions with the ferritin

molecules, these large defects were often scarlessly and

autonomously healed. In extreme cases, such as that shown

in Figure 6d and Video S4, the rhombohedral PIX could even

undergo near-complete lamellar fracturing and accordion-like

) they can be used to control the directionality of PIX

dynamics.

Anisotropic Mechanical and Self-Healing Properties

RAFT

of Rhombohedral

Ferritin PIX. Analogous to the

mechanical anisotropy of muscles enabled by their underlying

anisotropic architecture, the directional alignment of polymer

chains or embedded particles within hydrogels have been

shown to yield anisotropic mechanical properties with respect

to the direction of applied force and generate bending

motions.

rhombohedral

4,35

This behavior was also borne out in expanded

RAFT

ferritin PIX, which possess an alternating

motions to adapt to Ca ﬂuxes in solution, followed by full

pattern of high- and low-molecular density regions aligned

recovery of their original polyhedral morphology within

seconds. Such rapid, adaptive motions with attendant self-

healing are more typical of soft biological devices like muscles

rather than stiﬀ molecular crystals.

along the c-axis (Figure 6). When the expanded PIX were

exposed to Ca ions to induce contraction, they underwent a

drastic bending motion toward the direction of Ca inﬂux,

with ﬂexion angles of up ∼25° in the absence of any apparent

CONCLUSIONS

cracking. The PIX reverted to the original shape as the Ca

■

ﬂux dissipated (Figure 6a and Video S4). The bending of the

Ranging from abalone nacre and mussel byssus to spider silk

and skeletal muscles, nature uses the hierarchical assembly of

multicomponent materials to simultaneously achieve a

combination of vital properties (e.g., strength, toughness,

ﬂexibility, damage tolerance) that would be impossible to

obtain through the self-assembly of single components

PIX arises from the compression of the hydrogel matrix

perpendicular to the hexagonal ferritin ab-layers at the Ca

diﬀusion front and provides, in essence, a chemosensory/

chemotactic motion (Figure 6b). The actuation is remarkably

−

rapid with a bending rate of >10° s , which is one-to-several

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society

https://pubs.acs.org/10.1021/jacs.0c10065

Article

1,52

alone.

Accordingly, we have shown here that through the

Jake B. Bailey − Department of Chemistry and Biochemistry,

University of California, San Diego, La Jolla, California 92093,

United States

Ling Zhang − Department of Chemistry and Biochemistry,

University of California, San Diego, La Jolla, California 92093,

United States

physical integration of two disparate classes of materials, i.e.,

molecular crystals and hydrogel polymers, we can obtain an

unprecedented combination of material attributes and

mechanical behaviors: atomic-level order/coherence, direc-

tional motion, ﬂexibility, rapid anisotropic actuation, chemical

responsiveness, self-healing.

Complete contact information is available at:

Key to the attainment of anisotropic properties in PIX was

the ability of ferritin molecules to form lattices with distinct

symmetries and protein−protein interfaces. These diﬀerences

allowed the templation of alternatively patterned hydrogel

networks in situ, which ultimately enabled ferritin crystals that

essentially possess the same macroscopic morphologies to

display orthogonally directed motions. The original intent of

this study was to achieve control over the spatial distribution of

polymer networks within protein crystals using site-directed

RAFT-polymerization strategies. Although our ﬁndings

revealed that such strategies were not necessary to create

patterned hydrogels in crystallo, we posit they would still oﬀer

important advantages if their eﬃciencies can be improved, such

as the incorporation of polymers with a diverse range of

functional groups into protein lattices (regardless of their

chemical compatibility with the protein components),

construction of multipolymer networks, and spatiotemporal

control over polymer growth within lattices. Combined with

the inherent chemical versatility and functions of proteins, such

covalently hybridized PIX could oﬀer a unique platform for the

study of protein−polymer interactions and the development of

biocatalytic and molecular encapsulation/delivery systems with

tunable and responsive mechanical properties.

Notes

The authors declare no competing ﬁnancial interest.

Coordinate and structure factor ﬁles have been deposited into

the RCSB databank under the following PDB IDs: 6WYF,

RAFT

cubic

ferritin; 6WYG, rhombohedral

ferritin; 6WYH,

RAFT

Ac-infused rhombohedral

ferritin; 7K26, Ac-infused

ΔC

trigonal ferritin.

ACKNOWLEDGMENTS

■

We thank the following colleagues for assistance: R. Alberstein

and R. Subramanian for data processing and helpful

discussions; A. Rheingold, H. Nguyen, and M. Gembicky for

XRD; I. Rajkovic for SAXS; W.J. Rappel for confocal

microscopy. This work was funded by the US Army Research

Oﬃce (W911NF-19-1-0228) for materials design and

structural/mechanical characterization. Additional funding

was provided by US Department of Energy (BES, Division

of Materials Sciences, Biomolecular Materials Program, DE-

SC0003844) for SAXS studies and the initial development of

the PIX concept. Crystallographic data were collected at

Stanford Synchrotron Radiation Lightsource (SSRL), Ad-

vanced Light Source (ALS) and the Crystallography Facility of

the University of California, San Diego. SAXS data were

collected at SSRL. SSRL and ALS are supported by the DOE

Oﬃce of Science, Oﬃce of Basic Energy Sciences under

contracts DE-AC02-76SF00515 and DE-AC02-05CH11231,

respectively.

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/jacs.0c10065.

■

PDF )

In-crystallo formation of the hydrogel network moni-

tored by pyranine quenching (MOV )

Structural dynamics of a rhombohedral ^R^A^F^Tferritin PIX

during polymerization, expansion, and contraction

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