Organized arrangement of calcium carbonate crystals, directed by a

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Organized arrangement of calcium carbonate crystals, directed by a rationally designed protein Hang Ping, Yamin Wan, Hao Xie, Jing jing Xie, Weimin Wang, Hao Wang, Zuhair A. Munir, and Zhengyi Fu Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00365 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Crystal Growth & Design

Cover page Organized arrangement of calcium carbonate crystals, directed by a rationally designed protein Hang Ping,† Yamin Wan,† Hao Xie,†,‡,* Jingjing Xie,† Weimin Wang,† Hao Wang,† Zuhair A. Munir,§ and Zhengyi Fu†,* † State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan, 430070, China ‡ School of Chemistry, Chemical Engineering, and Life Science, Wuhan University of Technology, Wuhan, 430070, China § Department of Materials Science and Engineering, University of California, Davis, CA 95616, USA

ABSTRACT The integration of “brick and mortar” structure and superior fracture toughness of nacre has attracted intensive attentions recently. Elucidating interactions between biomacromolecules and inorganic phases is beneficial to understand the structure-forming process of nacre. Herein, a recombinant protein, ChiSifiCa, mimicking features of organic matrix in nacre is rationally designed. This protein contains three functional domains, chitin binding (Chi), silk fibroin (Sifi) and calcium ion binding (Ca). The domain order in ChiSifiCa is in the same manner as the distribution of organic matrix in nacre. When ChiSifiCa binds to the surface of chitin, it provides a confined micro-environment facilitating CaCO3 mineralization. The formation of spherical vaterite minerals with hollow structure were observed under the function of ChiSifiCa. These minerals are assembled by well-aligned nanoplatelets and nanoparticles, distributed in the outer and inner region of sphere, respectively. The nanoparticles were orientated radially from the center to the edge. These nanoplatelets with stacked multilayers display almost the same crystallographic orientation. During the biomineralization process, there is change of secondary structure of ChiSifiCa from random coil to α-helix.

Corresponding author: Zhengyi Fu, E-mail: [email protected] Wuhan University of Technology, Wuhan 430070, China Tel.: +86 027 87662983; fax: +86 027 87879468

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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Organized

arrangement

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calcium

carbonate

crystals, directed by a rationally designed protein Hang Ping,† Yamin Wan,† Hao Xie,†,‡,* Jingjing Xie,† Weimin Wang,† Hao Wang,† Zuhair A. Munir,§ and Zhengyi Fu†,* † State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan, 430070, China ‡ School of Chemistry, Chemical Engineering, and Life Science, Wuhan University of Technology, Wuhan, 430070, China § Department of Materials Science and Engineering, University of California, Davis, CA 95616, USA KEYWORDS: Multi-functional protein, rational design, biomineralization, organized arrangement, calcium carbonate

ABSTRACT The integration of “brick and mortar” structure and superior fracture toughness of nacre has attracted intensive attentions recently. Elucidating interactions between biomacromolecules and inorganic phases is beneficial to understand the structure-forming process of nacre. Herein, a recombinant protein, ChiSifiCa, mimicking features of organic matrix in nacre is rationally designed. This protein contains three functional domains, chitin binding (Chi), silk fibroin (Sifi)


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and calcium ion binding (Ca). The domain order in ChiSifiCa is in the same manner as the distribution of organic matrix in nacre. When ChiSifiCa binds to the surface of chitin, it provides a confined micro-environment facilitating CaCO3 mineralization. The formation of spherical vaterite minerals with hollow structure were observed under the function of ChiSifiCa. These minerals are assembled by well-aligned nanoplatelets and nanoparticles, distributed in the outer and inner region of sphere, respectively. The nanoparticles were orientated radially from the center to the edge. These nanoplatelets with stacked multilayers display almost the same crystallographic orientation. During the biomineralization process, there is change of secondary structure of ChiSifiCa from random coil to α-helix.

1. INTRODUCTION Biological systems are amazed by their unique bio-processing and bio-structures. The concept of “bioprocess-inspired fabrication” is put forward through learning the structure forming process of bio-systems in nature.1-4 The aim is to develop fabrication techniques for synthesizing and processing materials with novel structures and functions. A biological process involves a set of biochemical reactions under temporal and spatial regulations.5 Biomineralization is a typical biological process that produces minerals in a confined space under the functions of biomacromolecules. Biomacromolecules and confined space synergistically control the morphology, polymorph and orientation of biominerals.6,7 For example, non-collagenous proteins adsorbing in the gap zones of collagen fibrils can regulate the oriented growth of apatite inside collagen fibrils.3 Acidic proteins and silk fibroin fill inside lamellar layers of chitin and direct the growth of aragonite crystals.8


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Nacre is an intensively researched biomineral for its unique “brick and mortar” structure and superior mechanical properties.9,10 The major organic components of nacre include β-chitin matrix, hydrophobic silk fibroin, and hydrophilic acidic proteins. It has been verified that the chitin matrix is in the β-form with parallel chains and acts as a framework to direct the orientation of CaCO3.11-12 The oriented chitin film could guide the growth of calcite rods, assembled by rhombohedral nanocrystals with ordered arrangement.11 Silk fibroin is hydrophobic owing to the high contents of Gly and Ala amino acid residues. It serves as a space filler between the chitin sheets and controls the crystallization of aragonite crystals.13,14 The size and morphology of aragonite were precisely tuned by modifying the concentration of calcium ions and silk fibroin.14 The acidic proteins are distributed in silk fibroin gel and regulate the morphology and lattice structure of CaCO3 through functional domains, active acidic groups.15,16 Owing to the interaction between carboxyl group and calcium ions, it is easy for acidic proteins being adsorbed on specific surface of crystals and affecting their growth pathways, or being occluded inside crystals and disrupting the periodic structure.15,16 However, the formation of nacre is complicated in vivo, involving synergistic effects among multiple components. Current studies mainly focused on a single component to induce CaCO3 mineralization. It is necessary to develop systems that consolidated functional proteins and twodimensionally confined chitin matrix to reveal the formation of calcium carbonate. The aragonite-binding protein (Pif80-22) could induce the growth of aragonite on chitin surface, while only calcite phase was observed in the absence of chitin.17 The recombinant acidic proteins (rCAP-1), including chitin binding domain and C-terminal acidic region, promoted the oriented growth of CaCO3 crystals on a chitin matrix.18 Recently, we constructed a multifunctional


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protein ChiCaSifi (containing domains of chitin binding, calcium ion binding and silk fibroin) and anchored it on chitin surface to direct vaterite mineralization.19 The general model of nacre formation was described by Lia Addadi.8 The microenvironment is formed by two layers of β-chitin, with a gel comprising silk-like protein filling the space. Acidic proteins are adsorbed on chitin surface and act as active sites for the mineral growth. To gain deep insight into nacre formation, it is essential to investigate the effects of recombinant protein with different functional domains to regulate CaCO3 mineralization in a confined space. Therefore, we tend to construct recombinant proteins, ChiSifiCa and ChiCaSifi, and initially mimic the microenvironment in nacre, and regulate the growth of calcium carbonate on the surface of chitin. In the present study, we changed the domain order of the previously constructed protein ChiCaSifi19 and rationally designed a protein ChiSifiCa by putting the silk fibroin domain (Sifi) between the chitin binding domain (Chi) and the calcium ion binding domain (Ca). The domain order in ChiSifiCa is similar to the distribution of organic matrix in nacre. The ChiSifiCa can bind with the chitin resin through chitin binding domain. Silk fibroin domain and calcium ion binding domain can direct the nucleation and growth of CaCO3 crystals. The expression and purification of ChiSifiCa were attempted. The biological activity of ChiSifiCa in binding with chitin and calcium ions was investigated. Effects of ChiSifiCa were explored to direct vaterite growth on the surface of chitin matrix. 2. EXPERIMENTAL SECTION 2.1 Expression and purification of recombinant protein The coding sequence of the silk fibroin protein and the calcium binding domain were synthesized and inserted into the plasmid pTWIN1 (New England Biolabs, USA) downstream of


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the chitin binding domain. The resultant plasmid is pTWIN(ChiSifiCa) harboring the gene of ChiSifiCa. All constructs were confirmed by DNA sequencing. The plasmid pTWIN(ChiSifiCa) was transformed into E. coli BL21(ER2566). Expression and purification of protein ChiSifiCa was based on the IMPACTTM-TWIN user manual (New England Biolabs, USA). A single colony of E. coli BL21(ER2566) harboring pTWIN(ChiSifiCa) was inoculated into Luria-Bertani (LB) medium containing ampicillin (0.1 mg mL-1) and shaken with 200 rpm at 37 °C overnight. The cell suspension was cultured into LB medium by shaking at 37 °C until an OD600 of 0.5-0.6 was reached. Protein expression was induced by supplying with 1 mM IPTG and shaking continuously for 3 hours at 30 °C. Cells were harvested by centrifugation at 6,000 g at 4 °C for 10 minutes, re-suspended in buffer A (20 mM Tris-HCl, pH 8.5; 500 Mm NaCl; 0.2 % Tween 20; 1 mM EDTA), and lysed using a French press. The cell lysate was centrifuged at 12,000 g at 4 °C for 30 minutes. The supernatant was subjected to chitin affinity chromatography. Unbound proteins were washed with buffer B (20 mM Tris-HCl, pH 8.5; 500 mM NaCl; 1 mM EDTA). The bound ChiSifiCa was eluted with 1% sodium dodecyl sulfonate (SDS) or 6 M GuHCl and dialyzed successively with buffer C (20 mM Tris-HCl, pH 8.5; 500 mM NaCl; 0.2% Tween 20) for 2 hours, buffer D (20 mM Tris-HCl, pH 8.5; 500 mM NaCl; 0.1% Tween 20) for another 2 hours, and physiological saline (0.9% NaCl) for 8 hours. 2.2 Biological activity of recombinant protein The secondary structure of ChiSifiCa was measured by circular dichroism (CD) in an AVIV 60 CD spectrometer using 1 nm bandwidth and a scanning rate of 20 nm min-1. The binding activity of recombinant protein to chitin resin was determined by measuring the UV-Vis absorption. The equilibrated chitin resin was mixed with 100 µg recombinant protein solution overnight by gentle


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shaking at 4 °C. The supernatant was tested by UV-Vis spectrophotometer UV-2550 between 240 nm to 300 nm. The calcium ions binding activity of ChiSifiCa was determined by the intrinsic fluorescence change of ChiSifiCa titrating with calcium ions. Fluorescence emission of ChiSifiCa was recorded between 310 nm and 460 nm with an excitation wavelength at 295 nm. 2.3 Mineralization of CaCO3 in vitro The chitin resin was firstly mixed with purified ChiSifiCa, then CaCl2 (Sinopharm, China) was added to the above solution at a final concentration of 10 mM. The mineralization was initiated by adding 10 mM NaHCO3 (Sinopharm, China) at room temperature. The pH of solution was adjusted to 8.5. The chitin resin along with CaCO3 minerals was collected by centrifugation at 1,000 g for 5 minutes and washed with deionized water for three times. In the absence of chitin, NaHCO3 was directly added into ChiSifiCa and CaCl2 solution with the same condition. In the presence of physiological saline, the concentration of NaCl in solution is 0.9 wt%. In the control group of BSA, the reaction condition is the same to ChiSifiCa. 2.4 Characterization of mineralized products The morphology of CaCO3 minerals was provided by field emission scanning electron microscopy (FESEM) using a Hitachi S-4800 at 5 kV. The phase composition and crystallinity were examined by X-ray diffraction (XRD) using a Bruker D8 Advance, equipped with Cu Kα radiation (V = 40 kV, I = 40 mA). The diffraction patterns were collected in the range of 20-60 degree, with a scan step of 0.5 degree/minute. The secondary structure of recombinant protein was evaluated with Fourier transform infrared spectroscopy (FTIR) using a ThermoScientific Nicolet 6700. The FTIR spectra were collected from 4000 to 400 cm-1, at a resolution of 4 cm-1 with 32 scans. The fine structure of minerals was investigated with high-resolution transmission electron microscopy (HRTEM) on a JEM 2100F at 200 kV. The phase identification and


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crystallographic orientation of crystals was obtained by using selected area electron diffraction (SAED). 3. RESULTS AND DISCUSSION 3.1 Purification and biological activity of recombinant protein In order to simulate the spatial distribution of the organic matrix in nacre, the silk fibroin domain was inserted between the chitin binding domain and the calcium ion binding domain at the genetic level (Fig. 1a). The sequence of silk fibroin is composed of a repeated amino acid motif GAGAGS, which can fine control the size and morphology of calcium carbonate (Fig. 1b).14 The chitin binding domain is from the plasmid pTWIN1 itself, silk fibroin and calcium ion binding domain are fused into its C-terminus. It serves as an affinity tag for binding with chitin beads to facilitate purification as well as anchoring the recombinant protein ChiSifiCa on the chitin surface.20 The calcium ion binding domain stems from matrix protein MSI7, its DGD site is crucial for the growth of aragonite.21 The recombinant protein ChiSifiCa was overexpressed in the prokaryotic host E. coli (Fig. 1c). Purification of ChiSifiCa was achieved after stringent elution. SDS-PAGE analysis showed that the observed molecular weight of the ChiSifiCa matched well with the theoretical one (Fig. 1c). The purified of ChiSifiCa had over 95% homogeneity. Typically, the protein yield can be expected to 10 mg L-1 culture medium.


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Figure 1. Design, expression and purification of recombinant protein ChiSifiCa. (a) Scheme of the plasmid vector of pTWIN(ChiSifiCa). (b) The amino acid sequence of ChiSifiCa. The Chi domain, Sifi domain, and Ca domain are highlighted in red, black, and blue, respectively. (c) Expression and purification of ChiSifiCa, analysed with 15% SDS-PAGE. Lane 1, molecular weight maker; lane 2 and 3, lysate of uninduced and IPTG-induced cells, respectively; lane 4, affinity purified ChiSifiCa. The ChiSifiCa protein band is pointed by a black arrow. The secondary structure of purified ChiSifiCa was analysed by circular dichroism (CD) spectroscopy. A ellipticity band near 200 nm-1 indicates that the dominant secondary structure of ChiSifiCa is random coil (Fig. 2a). Specific interaction between ChiSifiCa and chitin resins was investigated through UV-Vis spectrum. The purified protein solution showed a strong absorption peak at 280 nm, which was attributed to the conjugated double bond between tryptophan (Trp) and tyrosine (Tyr) amino acids (Fig. 2b).22 When the protein solution was mixed with chitin resins, the supernatant did not show any absorption peak at 280 nm. Similarly, there was no absorption peak after washing the chitin resins. SDS-PAGE analysis proved that ChiSifiCa is the only protein that was bound with chitin resins (Fig. S1). This is due to the strong specific interaction between the chitin binding domain of ChiSifiCa and the chitin resins through a


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hydrophobic interaction. The calcium ions binding capacity of ChiSifiCa was measured through the intrinsic fluorescence changes (Fig. 2c). The fluorescence intensity at 346.5 nm gradually decreased when the concentration of calcium ions was increased. The fluorescence quenching constant was calculated through the Stokes-Einstein equation (Fig. 2d).23 The binding constant was 6.3, implying the specific binding with calcium ions.

Figure 2. Biological activity of ChiSifiCa. (a) CD spectra of purified ChiSifiCa. (b) The UV-Vis spectrum of protein solution (red curve), the supernatant after mixing with chitin resins (blue curve), the solution after washing protein bonded chitin resins (black curve). (c) Fluorescence change upon calcium titration with ChiSifiCa. (d) Linear fitting of fluorescence intensity F at 346.5 nm against the calcium concentration. 3.2 CaCO3 mineralization under the function of ChiSifiCa Mineralization of CaCO3 under the function of ChiSifiCa was investigated on the surface of chitin. Anchoring ChiSifiCa on the chitin surface can provide a confined micro-environment to facilitate CaCO3 nucleation and growth. Deposition of irregular shuttle-like minerals was


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observed in 1 minute on chitin surface treated with 20 µg/ml of ChiSifiCa (Fig. 3a). The shuttlelike minerals were assembled by nanoparticles (Fig. 3b). The alignment of interior nanoparticles was well organized, as shown by the direction of arrow. While, some holes appeared in the center of minerals, indicating that the interior nanoparticles were dissolved. The size of minerals and holes increased to micrometer scale, and the morphology evaluated to semi-sphere in 5 minutes of mineralization (Fig. 3c). There was no diffraction peak of the minerals in 1 minute and 5 minutes of mineralization, which was ascribed to the low content of minerals on chitin surface (Fig. 4). In 30 minutes of reaction, the hole was gradually closed through the stacking of nanocrystals on the top of the semi-spherical mineral, and the round-shaped minerals were obtained (Fig. 3d). The morphology of crystals near the edge was gradually evolved into quasi-hexagonal nanoplatelets (Fig. S2a-b). In the view from broken mineral, the nanoparticles were orientated radially from the center to the edge (Fig. S2c-d). The crystallographic phase was indexed to vaterite (JCPDS no. 33-0268) by XRD analysis (Fig. 4).


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Figure 3. SEM images of CaCO3 minerals on the surface of chitin. (a) Low and (b) high magnification of minerals obtained in 1 minute of mineralization, (c) 5 minutes, (d) 30 minutes, (e) 60 minutes. (e, inset) The equator of mineral. Scale bar in the inset is 200 nm. The equator of the minerals was pointed by red arrows. (f) Minerals obtained in the absence of protein. The intact sphere of minerals appeared, and the hole almost disappeared in 60 minutes of mineralization (Fig. 3e). In the equator of the minerals, the assembly of nanoplatelets was stacked multilayers from a side view (Fig. 3e inset). TEM image shows a hollow structure of mineral (Fig. 5a). In the red region, the single crystal-like SAED pattern confirms the same orientation of nanoplatelets (Fig. 5b). The arc-shaped pattern in the blue region indicates nanoparticles with slightly distorted orientation (Fig. 5c). These SAED patterns confirm the high crystallinity of the microstructures. The distribution of nanoparticles and nanoplatelets was also verified in a broken sphere (Fig. 5d). The same growth orientation of neighbouring two nanoplatelets under different layers was also evidenced (Fig. 5e). The crystal lattice, with an


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interplanar spacing of 0.357 nm, is parallel to the (110) plane of vaterite. The amorphous layer with about 5 nm thickness around the crystalline region was observed, which means that the growth of vaterite crystals may involve the transformation from amorphous phase. However, the regular calcite crystals are observed on the chitin surface in the absence of ChiSifiCa (Fig. 3f and Fig. S3). When the reaction time was prolonged to 5 hours, the intact sphere structure was maintained. The nanoplatelets-like crystals were merged into large disc-like crystals (Fig. S4a-b). After the mineralization of 12 hours, the order degree of nanocrystals in the equator was decreased, some crystals on the upside of sphere were gradually evolved into single crystal (Fig. S4c-d). The crystalline phase was changed from vaterite into calcite (Fig. S4e). In addition, the effect of physiological saline (0.9 wt% NaCl) and BSA were verified after the 60 minutes’ mineralization. In the presence of NaCl, rhombohedral calcite crystals deposited on chitin surface (Fig. S5a-b). Irregular morphology of crystals with mixed calcite and vaterite phases aggregated together under the influence of BSA (Fig. S5c-e). And minerals with a large size distribution and mixed phases were obtained under the effects of ChiSifiCa in the absence of chitin (Fig. S6). The irregular arrangement of nanocrystals on the surface was observed (Fig. S6 inset). It demonstrates that the synergistic effects between ChiSifiCa and chitin are crucial for the growth of vaterite minerals with organized nanocrystals. At the initial stage of mineralization in the ChiSifiCa system, the shuttle-like minerals are assembled by well-organized nanoparticles on the surface of chitin. The chitin surface provides two-dimensional confinement for the arrangement of nanoparticles. Based on this stacking behaviour, the minerals with multilayered structure and the building blocks with orientated growth are observed at the final stage. In previous work, the minerals consist of nanoparticles with random distribution in the ChiCaSifi system.19 We propose


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that the different micro-environments provided by proteins were the cause of these different observations. However, the detailed difference of function between ChiCaSifi and ChiSifiCa need further explore in the future.

Figure 4. XRD patterns of CaCO3 minerals acquired at various reaction time. 3.3 Roles of ChiSifiCa in CaCO3 mineralization Previous studies have shown that biomineral-associated proteins can template or catalyse the biomineralization.24,25 Structural changes of mineral proteins were observed during the biomineralization process.19,26,27 In the present study, we explored the CaCO3 biomineralization as well as changes of secondary structure of ChiSifiCa with the time course evolution by means of FTIR (Fig. 6a). The characteristic peaks of FTIR at 1425 cm-1, 1075 cm-1 and 863 cm-1 corresponded to the carbonate group of asymmetric stretching vibration (ν3), the symmetric stretching vibration (ν1), and the out-of-plane bending vibration (ν2), respectively.28,29 The


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increase of the intensity of the characteristic peak of vaterite at 747 cm-1 shows the enhanced content of CaCO3. The high-resolution of absorbance spectra between 1600 cm-1 and 1680 cm-1 were deconvoluted into three components corresponding to β-sheet, random coil and α-helix (Fig. 6b and Fig. S7). The chitin binding domain was predicted to form β-sheet structure and did not involve the mineralization of calcium carbonate.19 The area of α-helix increased along with the reaction time, and the area of random coil decreased (Table S1). This implies that the silk fibroin domain may change from random coil to α-helix during the process of CaCO3 crystallization.28 The change of secondary structure was also observed in ChiCaSifi directed CaCO3 mineralization; it can stabilize the vaterite phase of the minerals.19,31

Figure 5. Characterization of CaCO3 minerals after 60 minutes of mineralization. (a) TEM image of spherical mineral. (b) and (c) SAED patterns of corresponding red and blue region in(a). (d) TEM image of a broken sphere. (e) High resolution TEM of nanoplatelets in red arrow indication in (d).


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The amount of proteins can significantly affect the biomineralization. The CaCO3 mineralization as a function of different amount of ChiSifiCa was studied on the surface of chitin. SEM observations revealed that spindle-like minerals were obtained when 5 µg/ml ChiSifiCa presented in the mineralized system (Fig. S8a). Nanoparticles were randomly distributed at the equator. The product contained two phases: calcite and vaterite (Fig. S9). The reason for this may be that the amount of protein was not sufficient to stabilize the vaterite. Some vaterite had transformed to calcite. When the ChiSifiCa concentration increased to 10 µg/ml, only vaterite was contained in the spherical product (Fig. S8b). No calcite was observed. When the ChiSifiCa concentration was increased to 20 µg/ml, uniform spherical minerals were obtained (Fig. S8c). The regular stacking structure at the equator was observed. When the ChiSifiCa concentration was increased to 30 µg/ml, deposition of irregular disc-like vaterite minerals was observed on the chitin surface (Fig. S8d). It is possible that the high concentration of ChiSifiCa caused a curtailment of the growth of the minerals. The mineralization of CaCO3 at different pH levels was examined in the presence of 20 µg/ml ChiSifiCa. At pH 7.5, the growth of CaCO3 minerals was prohibited in the weak basic environment. CaCO3 particles aggregated and did not form spherical shapes (Fig. S10a-b). At pH 9.5, the conversion of HCO3- to CO32- was promoted. At the site of mineralization, the rise of CO32- concentration may enhance the nucleation rate of CaCO3.32 Therefore, the growth of nanoparticles was uncontrollable, and non-uniform size of minerals deposited on chitin (Fig. S10c). Nanoparticles with different sizes were observed on the surface of spherical mineral (Fig. S10d). XRD showed that the resulting CaCO3 was a mixed phase of calcite and vaterite (Fig. S11).


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There have been reports that different polyelectrolytes and block co-polymers were used as crystal growth modifiers to prepared calcium carbonate with hollow structures or layered structures. For example, Double-hydrophilic block copolymers (DHBCs) are frequently designed to control the mineralization reactions through their hydrophilic building blocks. One block interacts with inorganic minerals, and a non-interacting block induces the assembly of mineral units into macroscopic superstructures.33 The controlled surface structure, shape and size of CaCO3 spherules were synthesized by using PEG-b-PEIPA or PEG-b-PMAA.34 Various metal carbonate systems with well-defined structures, such as CaCO3, BaCO3, CdCO3, MnCO3, and PbCO3, were prepared by using a set of DHBCs as crystal modifiers. The selective interaction between the functional groups of polymers and crystals were determined by electrostatic interaction.35 PEG-b-hexacyclen was exploited to direct the formation of unusual calcite pancakes with multiple stacked layers. The formation of new layers on mother layer was in a step-wise manner.36 Block copolymer PEG-b-pGlu was adopted to controlled synthesis of highly monodisperse vaterite microspheres in a suitable mixture of solvents through a gas-liquid diffusion method.37 Inspired from the functions of DHBCs, the formation processes of CaCO3 under the effect of recombinant protein were proposed.


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Figure 6. Time-dependent FTIR measurements of CaCO3 minerals at various reaction time. (a) The transmittance spectra of minerals. (b) The high-resolution of absorbance spectra of the pink area in (a). The process of forming CaCO3 directed by ChiSifiCa on the surface of chitin was illustrated in Figure 7. First, the recombinant protein anchored on the surface of chitin and sequestered calcium ions from solution, then served as nucleation sites for the formation of nanocrystals due to the high localized concentration of calcium ions.38 The alignment of aggregated vaterite nanoparticles may be directed by the structural change of protein during crystal formation. The


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protein ChiSifiCa not only modulate the mineral crystalline phase to vaterite, but also controls the orientation of nanoparticles in the confined chitin surface. Like that of ChiCaSifi, the change of protein secondary structure of ChiSifiCa from random coil to α-helix also makes contributions to the CaCO3 mineralization process. The formation of hollow structure was driven by the dissolution of interior nanocrystals toward the outside.38 Because the hybrid core was composed of aggregated nanoparticles with a large amount of protein, which should have a tendency to dissolve the central nanocrystals. Subsequently, the nanoplatelets-like crystals formed at the periphery of the sphere structure. The heterogeneous texture with an outward-oriented ascribed to the secondary nucleation in solution with decreasing protein concentration gradient. The nanoplatelets stacked multilayers on the base of the above structure. The minerals were spherical, and the structure of nanoplatelets at the equator of the mineral was multilayers.

Figure 7. Schematic of CaCO3 mineralization directed by ChiSifiCa on the surface of chitin. 4. CONCLUSIONS The recombinant protein ChiSifiCa was rationally designed to simulate the spatial distribution of natural organic matrix in nacre. Anchoring ChiSifiCa on the surface of chitin provided a confined micro-environment to regulate calcium carbonate mineralization. The spherical vaterite


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with hollow structure, assembled by nanocrystals, was deposited on the chitin surface. The nanoparticles were orientated radially from the center to the edge, and the marginal area was constructed by oriented nanoplatelets. Characteristic layered structure of minerals was observed at the equator of the minerals. During the biomineralization process, the secondary structure of ChiSifiCa transformed from random coil to α-helix, directly evidenced the involving of protein for mineral formation. The protein concentration and pH can affect the morphology and crystalline phase of CaCO3 minerals. This study provides a clearer insight into biomineralization by recombinant proteins, and also opens a novel path for fabrication materials with new structures and functions.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI . SDS-PAGE analysis of bioactivity of recombinant protein. SEM images of CaCO3 minerals acquired at different conditions. SEM images and XRD patterns of control groups. SEM images and XRD patterns of CaCO3 minerals acquired at longer reaction time. FTIR spectra of CaCO3 minerals at various reaction time. XRD patterns of CaCO3 minerals at different concentration. SEM images and XRD patterns of CaCO3 minerals at different pH.

AUTHOR INFORMATION Corresponding Author


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*E-mail: [email protected] (Zhengyi Fu) *E-mail: [email protected] (Hao Xie) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (51521001, 31771032), awarded by the National Natural Science Foundation of China. We acknowledge Miss Tingting Luo (Center for Materials Research and Analysis of Wuhan University of Technology) for the help in HRTEM analysis. We are grateful to Miss Bi-Chao Xu of the Core Facility and Technical Support, Wuhan Institute of Virology for her technical support in sample preparation. REFERENCES 1.

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For Table of Contents Use Only Organized arrangement of calcium carbonate crystals, directed by a rationally designed protein Hang Ping,† Yamin Wan,† Hao Xie,†,‡,* Jingjing Xie,† Weimin Wang,† Hao Wang,† Zuhair A. Munir,§ and Zhengyi Fu†,* † State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan, 430070, China ‡ School of Chemistry, Chemical Engineering, and Life Science, Wuhan University of Technology, Wuhan, 430070, China § Department of Materials Science and Engineering, University of California, Davis, CA 95616, USA

The recombinant protein ChiSifiCa was rationally designed to simulate the spatial distribution of natural organic matrix in nacre. Anchoring ChiSifiCa on the surface of chitin provided a confined micro-environment to regulate calcium carbonate mineralization. The spherical vaterite with hollow structure, assembled by organized nanocrystals, was deposited on the chitin surface.


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Organized arrangement of calcium carbonate crystals, directed by a

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