Biomimetic Synthesis of Aragonite Nanorod Aggregates with Unusual

Article pubs.acs.org/crystal

Biomimetic Synthesis of Aragonite Nanorod Aggregates with Unusual Morphologies Using a Novel Template of Natural Fibrous Proteins at Ambient Condition Zengqiong Huang and Gangsheng Zhang* College of Materials Science and Engineering, Guangxi University, Nanning, Guangxi 530004, China ABSTRACT: Biomimetic synthesis of calcium carbonate has been studied for decades in order to understand the biomineralization process. However, to date, it is still difficult to obtain pure aragonite by transformation of amorphous calcium carbonate (ACC) at room temperature. Herein, we obtained almost pure aragonite nanorod aggregates (ANA) with unusual morphologies via transformation of ACC at ambient conditions using a novel fibrous protein K58 from bivalve Siliqua radiata ligament as the template. We found that nanorods are formed initially as ACC containing disordered aragonite nanocrystals, which then evolve into oriented attached nanocrystals coated with minor ACC, and finally into aragonite single crystals. Further analysis reveals K58 is homologous to type II cytoskeletal 1 and type I cytoskeletal 9, with many acidic amino acid residues. It should be the carboxyl groups of these residues, which have short-range order similar to aragonite lattice, control short−range order ACC formation, resulting in the preferential transformation of ACC into aragonite. This article provides some valuable information for better understanding of aragonite crystal growth and biomineralization process; it also highlights the role of biomacromolecule in the formation of aragonite fibers in bivalve ligaments. protein: aspein of pearl oyster shell and Mg2+ as additives.15 More recently, aragonite crystals were obtained on a nacre polypeptide n16N and β-chitin combining substrate.16 Although these researches obtained more or less aragonite crystals, and calcite or vaterite is easy to synthesize by transformation of ACC,17−19 it is still a great challenge for scientists to obtain pure aragonite crystals via transformation of ACC with an individual protein or without any additives at ambient condition. In addition, to our knowledge, most of previous researches are concentrated on shell proteins; none has been given to proteins from bivalve ligaments. Bivalve ligament is a calcified structure connecting two shell valves dorsally and functions to open the valves as abductor muscles relax.20 Structurally, almost all bivalve ligaments are composed of an outer protein and an inner aragonite fiber layer.21 This structure, with the latter sticking tightly to the former, implies ligament protein layers are likely to act as templates to control the formation of aragonite fibers. To confirm this hypothesis, a novel fibrous protein layer, K58 from Siliqua radiata ligament,22 was used as a substrate to induce CaCO3 formation (Figure 1). Very strikingly, we found K58 alone is sufficient to control the formation of almost pure ANA with unusual morphologies via transformation of ACC at ambient condition. We also found the structure of K58 plays a

1. INTRODUCTION Biomineral, especially the most ubiquitous calcium carbonate (CaCO3), has attracted continuous attention due to its delicate structure and excellent mechanical properties in living organisms. Of the three common polymorphs of CaCO3, i.e., calcite, aragonite, and vaterite, the thermodynamically most stable calcite and slightly stable aragonite are widespread in biological systems.1,2 For example, most of mollusk shells are composed of a calcite prismatic layer and an aragonite nacreous layer. The nacre, which is composed alternately of pure aragonite tablets and organic matrix, shows extraordinary mechanical properties.3 To reveal the formation mechanism of aragonite tablets in nacre or to obtain pure aragonite materials, increasing efforts have focused on synthesizing aragonite by chemical methods. Various organics, such as Langmuir monolayer,4−6 selfassembled monolayers,7−9 polysaccharides,10,11 and polymers,12−14 have been used as templates to induce CaCO3 formation because organic matrixes are considered to be the key factor controlling the biomineralization process. These researches provide some valuable information on how organic templates can affect mineral formation. In addition, detailed studies have been carried out to induce the growth of CaCO3 using proteins of mollusk shells as substrates or additives. For example, Falini et al. discovered a combination of β-chitin, silk fibrin, and glycoproteins extracted from nacre or prismatic layer can induce aragonite or calcite formation, respectively.2 Takeuchi et al. obtained aragonite and calcite using acidic © 2012 American Chemical Society

Received: September 29, 2011 Revised: February 28, 2012 Published: March 2, 2012 1816

dx.doi.org/10.1021/cg201293f | Cryst. Growth Des. 2012, 12, 1816−1822

Crystal Growth & Design

Article

Figure 1. Optical photos of S. radiata (a) and fibrous protein (b) separated from the ligament.

crucial role in the formation and stabilization of ACC as well as in the transformation of ACC into aragonite.

Figure 2. SDS−PAGE pattern of marker (lane 1) and protein sample (lane 2).

2. EXPERIMENTAL SECTION

at 37 °C for 16 h. Then, it was treated twice with 50% acetonitrile and 0.1% trifluoroacetic acid and centrifuged to obtain a peptide solution. The solution was dried under vacuum, dissolved in 0.1% formic acid, desalted on a C18 peptide trap with 0.2% formic acid, and separated by a reversed phase C18 column with a linear gradient of 0−50% mobile phase A (0.1% formic acid/84% acetonitrile) in mobile phase B (0.1% formic acid) over 60 min. Separated peptides were analyzed on an LTQ Velos dual-pressure linear ion trap mass spectrometer (Thermo Finnigan, CA) equipped with a microspray source. The mass spectrometer was operated in data−dependent mode with spray voltage of 3.4 kV at 200 °C and full scan mass range of 300−1800 Da. The 10 most intense ions in every full scan were selected for MS−MS analysis. The acquired spectra were searched against mollusk and human protein databases using the SEQUEST algorithm on Bioworks 3.2 software. Delta CN (≥0.1) and Xcorr (one charge ≥ 1.9, two charges ≥ 2.2, and three charges ≥ 3.75) were used as criteria for identification.

2.1. Preparation of Protein Substrates. S. radiata ligaments and K58s were isolated mechanically from shells and ligaments, respectively. After being decalcified with 3% (v/v) acetic acid, washed with deionized water, and air-dried, K58s were detected by FTIR to confirm the complete decalcification. Then, pure K58s were used as substrates for CaCO3 precipitation and prepared for liquid chromatography−tandem mass spectrometry (LC/MS−MS) analysis. 2.2. Synthesis of Crystals. We put K58s and coverslips (control substrates) on the bottom of different culture dishes for CaCO3 precipitation. Crystals were grown by the CO2-diffusion method,23 i.e., culture dishes containing 80 mL of 10 mM calcium chloride (CaCl2) and a beaker containing 10 mL sulfuric acid were placed in a closed desiccator beside a watch glass containing ammonium bicarbonate (NH4HCO3) powder. The experiment was performed continuously from 1 h to 1 week at ambient conditions. To observe the crystal growth process, we collected K58s and coverslips at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 1 week, respectively, rinsed them with deionized water, and air-dried. 2.3. Characterization. Crystals collected at different times were analyzed by FTIR (Nicolet 4700, Thermo Electron) with a resolution of 4 cm−1. Absorption intensity ratios of calcite (877 cm−1):aragonite (857 cm−1) of obtained spectra were calculated and projected onto the standard reference curve plotted with intensity ratios vs calcite concentration (Figure 1 in ref 24) to obtain composition of calcite and aragonite. Crystal morphology was observed using a Hitachi S-3400N SEM operating at 20 kV. For TEM observations, K58s containing crystals obtained at 2 and 12 h were treated with ultrasonic in absolute alcohol for 10 min, respectively. TEM and HRTEM observations as well as SAED analysis was performed on a JEOL JEM-2010 HRTEM operating at 200 kV. 2.4. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS−PAGE). K58s were ground into powder in liquid nitrogen and treated with 7 M urea containing 3% 2-mercaptoethanol and 0.5 M sodium hydroxide (NaOH) solution at 65 °C for 1 h. After being centrifuged, the residue was treated again with the same condition except NaOH concentration was diluted into 0.25 M. The solution was dialyzed again with deionized water for three days. Dialysate was concentrated, treated with a 2D Clean-Up Kit (GE Healthcare), redissolved in lysis buffer, and quantified by Bradford assay. Protein sample (12.2 μg) and marker (MW 14.4−94.0 kDa) were applied to SDS−PAGE on a 12% separating gel. After being silver-stained, gel band 2-1 (Figure 2) was excised for trypsin digestion. 2.5. In−Gel Trypsin Digestion and LC/MS−MS Analysis. The gel band was destained, dehydrated, and digested by 10 ng/μL trypsin

3. RESULTS 3.1. FTIR Analysis of Synthetic Crystals. Crystal phases were analyzed by FTIR. From the obtained spectra, we did not find the characteristic split absorption bands of ACC at about 1420 and 1474 cm−1 and the broad peak at 866 cm−1. Therefore, we provide the spectra only from 1000 to 600 cm−1. Figure 3a shows characteristic absorption peaks of aragonite at about 857, 713, and 700 cm−1, accompanied with weak peaks of calcite at about 877 cm−1, indicating the existence of trace amount of calcite; whereas, Figure 3b presents only characteristic peaks of calcite at about 877 and 713 cm−1.25 Composition analysis results (Table 1) indicate that crystals grown on

Figure 3. FTIR spectra of crystals grown on protein substrates (a) and on coverslips (b) at different crystallization time. 1817



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and that the morphologies develop from sheaf-like (Figure 4a) to dumbbell-shaped (Figure 4b,c) and bouquet-shaped (Figure 4d), and finally turn gradually into spherical ANA (Figure 4e− g). Meanwhile, the length of individual ANA increases from 10 to 22 μm. It should be noted that the spherical ANA formed at 24 h (Figure 4f) show no significant changes in size and in morphology for the next 6 days duration of experiment (Figure 4g). More notably, many pyramidal structures can be seen at the end of individual ANA at 1 to 4 h of growth (insets in Figure 4a−c). These pyramidal structures, aggregated by many fine particles and showing rough surfaces (Figure 5a), were replaced by a lot of smooth radiated fibers after 8 h of deposition (Figures 4d and 5b). It is likely these fibers are assembled initially by lots of fine particles. Different from the crystals deposited on protein substrates, those grown on coverslips show perfect rhombohedral calcite from 1 h to 1 week. A typical sample collected at 24 h was shown in Figure 4h. 3.3. TEM Observations. To obtain detailed information of the pyramidal structures and radiated fibers in Figure 5, TEM observations were performed. As shown in Figure 6a, a rough nanorod coming from ANA obtained at 2 h is aggregated by lots of nanoparticles, which tend to orient along the long axis of the rod. When observing the rod with HRTEM and analyzed by fast Fourier transform (FFT), surprisingly, several irregular− shaped nanocrystals with random crystallographic orientations are surrounded by ACC (Figure 6b−d), which suggests that they are likely transformed from ACC, as no organic additives were used in our experiment. However, why have we failed to find ACC by FTIR analysis (Figure 3a)? It is because ACC is difficult to detect when associated with a crystalline form of aragonite.26 In addition to the rough nanorod, a relatively smooth one was also found at this growth stage. It shows a dark well− defined crystalline core consisting of nanocrystals attaching in a spiral bead-shaped structure (Figure 6e), which seems they are trying to rotate to unify their crystallographic orientations. Interestingly, the core is sheathed by a bright layer more than 9 nm thick, and it gets more and more bright along the [001] growth direction, indicating less crystallization at the growth edge (Figure 6e). Its HRTEM image and SAED pattern, which scatters like a single crystal (Figure 6f,g), confirm nanocrystals in the core attached orderly with a same crystallographic orientation along the [001] direction. While FFT of the bright sheathed layer shows a halo characteristic of ACC (Figure 6h). It should be noted that lattice fringes near the interface of crystal and ACC are lighter than those at the dark region (black arrows in Figure 6f), which implies aragonite crystals are transformed from ACC. Compared with these ACC sheathed nanorods, the one obtained at 12 h is homogeneous with a smooth and perfect surface (Figure 6i). It has transformed into a mature crystal showing the same crystallographic orientation as that obtained at 2 h (Figure 6j,f) and presents perfect diffraction spots characteristic of aragonite single crystal, with trace amount of ACC embed in crystal at the edge (Figure 6k,l). These findings point to the original natures of aragonite nanorods from transformation of ACC and ANA with unusual morphologies from self−assembly of nanorods. 3.4. Identification of K58. Peptides from trypsin digested K58 were successfully separated (Figure 7) and identified (Tables 2 and 3) by LC/MS−MS. When searched against the mollusk protein database, and no proteins of bivalve mollusk

Table 1. Composition of Aragonite and Calcite Grown on Different Substrates at Different Times substrate

crystallization time

aragonite

calcite

fibrous protein

1h 2h 4h 8h 12 h 24 h 1 week 1 h−1 week

95% 100% 97% 98% 99% 99% 98% 0%

5% 0% 3% 2% 1% 1% 2% 100%

coverslip

protein substrates are almost pure aragonite, while those obtained on coverslips are pure calcite. 3.2. Morphology Observations. Figure 4a−g shows the morphologies and evolution of ANA obtained from protein substrates at different times. It is obvious that with an increase of crystallization time, the number of ANA increases sharply

Figure 4. SEM images of crystals grown on protein substrates at different crystallization time (a−g) and on coverslip for 24 h (h). (a) One hour, showing sheaf-like ANA; (b,c) 2 and 4 h, growing into dumbbell-shaped ANA; (d) 8 h, showing bouquet-shaped ANA with radiated fibers; (e−g) 12, 24 h, and 1 week, respectively, developing into spherical ANA with lots of radiated fibers; (h) showing typical rhombohedral calcite. Insets are corresponding magnified images. 1818



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Figure 5. High magnified SEM images: panels a and b correspond to insets in Figure 4b,e, respectively. (a) Deposit for 2 h, showing pyramidal structures assembled by lots of fine particles (as indicated by white arrows); (b) deposit for 12 h, showing a lot of smooth radiated fibers.

Figure 6. TEM observations of nanorods obtained at different growth stages. (a) TEM image of a rough nanorod comprising lots of nanoparticles obtained at 2 h; (b) HRTEM image of the box area in panel a showing nanocrystals with random orientations; (c,d) FFT of panel b and white circle area in panel b, respectively, indicating that disordered nanocrystals are surrounded by ACC; (e) TEM image of another nanorod obtained at 2 h showing a bead-shaped crystalline core sheathed by ACC, and the black arrow indicates the [001] growth direction; (f) HRTEM image of the box area in panel e, and the black arrows indicate lattice fringes at different regions; (g) SAED pattern of panel f showing aragonite single crystal spots, and the zone axis is [11̅ 0]; (h) FFT of black circle area in panel f showing a halo characteristic of ACC; (i) TEM image of a homogeneous nanorod obtained at 12 h; (j) HRTEM image of the box area in panel I showing that the rod is well crystallized; (k) SAED of panel j scatters aragonite single crystal pattern,and the zone axis is [10̅ 0]; (l) FFT of white circle area in panel j showing the same spots as that in panel k, accompanied with a small dim halo characteristic of ACC.

4. DISCUSSION

were matched. However, when searched against human protein database, K58 was identified as keratin type II cytoskeletal 1 (KRT 1) and type I cytoskeletal 9 (KRT 9), with 32 and 29 matching peptides as well as 46.6% and 63.1% sequence coverage, respectively, representing 100% confidence. This result indicates K58 is homologous to KRT 1 and KRT 9.

On the basis of the above investigations, we concluded that K58 alone, without any additives, is sufficient to control the formation of almost pure ANA with unusual morphologies via transformation of ACC at ambient conditions. However, how can K58 induce ACC formation and affect the polymorph of 1819



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Figure 7. LC/MS−MS chromatogram of product ions of trypsin digested K58.

Table 2. LC/MS−MS Result Match with Keratin Type II Cytoskeletal 1 (KRT 1) [Homo sapiens], NCBI Database ID gil119395750 MH+

matching peptidesa,b

MH+

matching peptidesb

946.0403 974.0937 1016.1076 1034.1027 1066.1464 1093.0904 1126.1986 1128.2812 1158.2641 1180.2927 1202.3861 1266.3823 1278.4794 1303.484 1317.4315 1341.4492

R.GENALKDAK.N K.IEISELNR.V K.DVDGAYM*TK.V R.TLLEGEESR.M K.AQYEDIAQK.S R.GSGGGSSGGSIGGR.G K.AEAESLYQSK.Y K.KDVDGAYMTK.V R.DYQELM*NTK.L K.YEELQITAGR.H R.LRSEIDNVKK.Q R.TNAENEFVTIK.K K.LALDLEIATYR.T R.SLDLDSIIAEVK.A K.NMQDM*VEDYR.N K.SKAEAESLYQSK.Y

1358.4798 1384.5189 1394.5552 1476.6163 1524.7675 1600.7558 1639.8368 1658.7539 1688.9508 1717.8196 1943.105 1995.1833 2185.381 2287.4686 2385.2799 2934.2531

K.LNDLEDALQQAK.E K.SLNNQFASFIDK.V R.TNAENEFVTIKK.D K.WELLQQVDTSTR.T R.LLRDYQELMNTK.L K.NKLNDLEDALQQAK.E K.SLNNQFASFIDKVR.F R.SGGGFSSGSAGIINYQR.R R.SLVNLGGSKSISISVAR.G K.QISNLQQSISDAEQR.G K.LNDLEDALQQAKEDLAR.L R.THNLEPYFESFINNLR.R K.NKLNDLEDALQQAKEDLAR.L K.AEAESLYQSKYEELQITAGR.H R.GGGGGGYGSGGSSYGSGGGSYGSGGGGGGGR.G R.FLEQQNQVLQTKWELLQQVDTSTR.T

a

M*, oxidized methionine. bThe distances between the carboxyl groups of the first and last amino acid residues in bold italic peptides are almost equal to aragonite lattice constant a (0.496 nm) or b (0.798 nm).

Table 3. LC/MS−MS Result Match with Keratin Type I Cytoskeletal 9 (KRT 9) [Homo sapiens], NCBI Database ID gil55956899 MH+


MH+


809.93 812.9803 898.0191 982.9776 1061.1713 1067.201 1082.1709 1158.2471 1191.3804 1233.274 1236.23 1308.5322 1316.4429 1587.6707 1792.7189

R.LASYLDK.V K.KGPAAIQK.N R.MTLDDFR.I R.FSSSGGGGGGGR.F K.TLLDIDNTR.M K.FEMEQNLR.Q K.STM*QELNSR.L R.QGVDADINGLR.Q R.QVLDNLTMEK.S R.SGGGGGGGLGSGGSIR.S R.FSSSSGYGGGSSR.V R.IKFEMEQNLR.Q K.DQIVDLTVGNNK.T K.VQALEEANNDLENK.I R.GGSGGSYGGGGSGGGYGGGSGSR.G

1839.0399 1853.0901 1967.2128 2096.3176 2188.4621 2204.4615 2316.635 2378.578 2511.5966 2706.7378 2755.0484 2904.1342 3394.5597 3225.0872

R.HGVQELEIELQSQLSK.K K.TLNDMRQEYEQLIAK.N R.HGVQELEIELQSQLSKK.A R.QEIECQNQEYSLLLSIK.M K.SDLEM*QYETLQEELMALK.K K.SDLEM*QYETLQEELM*ALK.K K.SDLEMQYETLQEELM*ALKK.N R.LASYLDKVQALEEANNDLENK.I K.EIETYHNLLEGGQEDFESSGAGK.I R.GGGGSFGYSYGGGSGGGFSASSLGGGFGGGSR.G R.YCGQLQM*IQEQISNLEAQITDVR.Q K.NYSPYYNTIDDLKDQIVDLTVGNNK.T R.KDIENQYETQITQIEHEVSSSGQEVQSSAK.E R.GGSGGSHGGGSGFGGESGGSYGGGEEASGSGGGYGGGSGK.S

a

M*, oxidized methionine. bThe distances between the carboxyl groups of the first and last amino acid residues in bold italic peptides are almost equal to aragonite lattice constant a (0.496 nm) or b (0.798 nm).

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Figure 8. Schematic diagram representing the possible formation process of ANA: (a) carboxyl groups on protein surface attract and chelate calcium ions; (b) ACC with short-range order resembling aragonite crystal is formed by the controlling of carboxyl groups with short-range order similar to aragonite lattice; (c) ACC transform into aragonite nanocrystals with random orientations; (d) a single nanorod with oriented attached nanocrystals in a same crystallographic orientation is formed by the growing and rotating of nanocrystals; (e) ACCs attach to the single nanorod and transform into more nanorods; (f−h) by continually attaching ACC, growing and fusing of nanocrystals, and aggregating nanorods in a 2D manner, ANA turns gradually from dumbbell-shaped into spherical morphologies with smooth radiated fibers.

crystal. Note that most of the carboxyl groups of the acidic amino acid residues in bold italic peptides (Tables 2 and 3) belong to the α-helix center rod domains of the respective protein.27 When they involve in α-helix structure where the distance of adjacent residues is 0.15 nm,31 the distances between the first and last carboxyl group of these residues (3 or 5 residues apart, 0.45 or 0.75 nm) are almost equal to aragonite lattice constants (a = 0.496 nm; b = 0.798 nm). That means these carboxyl groups, with short-range order similar to aragonite lattice induce the formation of ACC with shortrange order resembling aragonite crystal, resulting in the preferential transformation of ACC into aragonite nanocrystals (Figure 8b,c). These aragonite nanocrystals, which spread disorderly in ACC with random crystallographic orientations and high internal energy, grow gradually to fuse with each other (Figures 6b and 8c) and tend to lower the energy by arranging orderly. Consequently, they rotate to get the same crystallographic orientation with driving force coming from the decrease of internal energy and removing of surface energy,32,33 then an ACC sheathed single nanorod comprising oriented attached nanocrystals is formed (Figures 6e and 8d). This nanorod is similar to the ACC-coated aragonite single crystal obtained by Nassif3 who also found ACC-coated aragonite platelets in nacre.34 These findings indicate that the transformation of ACC into aragonite may be ubiquitous not only in living organisms but also in in vitro experiments. With an increase of crystallization time, the immature ACC sheathed nanorod turns gradually into a mature single crystal by continual transformation of ACC into aragonite (Figure 6e,i). Meanwhile, some other colloidal ACC attach to the immature nanorod by Brownian motion and form more nanorods in the same manner (Figure 8d,e). Finally, ANA with unusual morphologies are formed by continually attaching ACC,

CaCO3? We believe the primary structure of K58 and its assembly path play crucial roles. In our previous study,22 we have reported the secondary structure of K58. Here, by LC/MS−MS analysis, we found K58 is homologous to KRT 1 and KRT 9, two proteins of the keratin family. That means K58 is likely to take the same manner as keratins to assemble into protein fibers. Briefly, K58 is a heteropolymer similar to other keratins.27 It is assembled initially by two monomers homologous to KRT 1 and KRT 9, respectively, to form a heterodimer; then tetramers, octamers, and 16 polymers are formed, until they turn into mature 10 nm width filaments.27 Finally, these filaments assemble into protein fibers. It should be this particular assembly way and the primary structure that contains a high content of acidic amino acids (9.2%)22 inducing ACC formation and modulating its transformation into aragonite. Further analysis of the matching peptides in Tables 2 and 3 confirmed our inference. These peptides contain many acidic amino acid residues (D, Asp; or E, Glu) and constitute a part of the monomer of respective protein.27 Since protein fibers of K58 are assembled by lots of monomers, each fiber is sure to have many acidic amino acid residues, with their carboxyl groups spreading disorderly or orderly on the protein surface. These carboxyl groups, which provide negative charges to attract and chelate calcium ions, induce the formation of ACC (Figure 8a,b). However, how do these carboxyl groups modulate the transformation of ACC into aragonite? Many reports have demonstrated that ACC is an unstable transient precursor phase for crystalline polymorphs;28−30 it can transform easily into calcite or vaterite in vitro.17−19 Addadi et al.26 discovered some ACC have short-range order, which sometimes resemble the crystalline form into which they are going to transform. We speculate that the formed ACC, which was induced by carboxyl groups, may have short-range order resembling aragonite 1821



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(13) Ajikumar, P. K.; Lakshminarayanan, R.; Valiyaveettil, S. Cryst. Growth Des. 2004, 4, 331−335. (14) Shi, S.; Su, Z.; Wei, H.; Chen, X. J. Appl. Polym. Sci. 2010, 117, 3308−3314. (15) Takeuchi, T.; Sarashina, I.; Iijima, M.; Endo, K. FEBS. Lett. 2008, 582, 591−596. (16) Keene, E. C.; Evans, J. S.; Estroff, L. A. Cryst. Growth Des. 2010, 10, 1383−1389. (17) Pouget, E. M.; Bomans, P. H. H.; Goos, J. A. C. M.; Frederik, P. M.; With, G.; Sommerdijk, N. A. J. M. Science 2009, 323, 1455−1458. (18) Kim, Y. Y.; Hetherington, N. B. J.; Noel, E. H.; Kröger, R.; Charnock, J. M.; Christenson, H. K.; Meldrum, F. C. Angew. Chem., Int. Ed. 2011, 50, 12572−12577. (19) Wang, Y. W.; Kim, Y. Y.; Stephens, C. J.; Meldrum, F. C.; Christenson, H. K. Cryst. Growth Des. 2012, 12, 1212−1217. (20) Zengqiong, H.; Gangsheng, Z. Micron 2011, 42, 706−711. (21) Sartori, A. F.; Ball, A. D. J. Molluscan Stud. 2009, 75, 295−304. (22) Huang, Z.; Zhang, G. Biochemistry 2011, 76, 1227−1232. (23) Qiao, L.; Feng, Q.; Lu, S. Cryst. Growth Des. 2008, 8, 1509− 1514. (24) Compere, E. L.; Bates, J. M. Limnol. Oceanogr. 1973, 18, 326− 331. (25) Liu, R.; Xu, X.; Cai, Y.; Cai, A.; Pan, H.; Tang, R.; Cho, K. Cryst. Growth Des. 2009, 9, 3095−3099. (26) Addadi, L.; Raz, S.; Weiner, S. Adv. Mater. 2003, 15, 959−970. (27) Szeverenyi, I.; Cassidy, A. J.; Chung, C. W.; Lee, B. T. K.; Common, J. E. A.; Ogg, S. C.; Chen, H.; Sim, S. Y.; Goh, W. L. P.; Ng, K. W.; Simpson, J. A.; Chee, L. L.; Eng, G. H.; Li, B.; Lunny, D. P.; Chuon, D.; Venkatesh, A.; Khoo, K. H.; Irwin McLean, W. H.; Lim, Y. P.; Lane, E. B. Hum. Mutat. 2008, 29, 351−360. See http://www. interfil.org/intro.php. (28) Weiss, I. M.; Tuross, N.; Addadi, L.; Weiner, S. J. Exp. Zool. 2002, 293, 478−491. (29) Politi, Y.; Kalisman, Y. L.; Raz, S.; Wilt, F.; Addadi, L.; Weiner, S.; Sagi, I. Adv. Funct. Mater. 2006, 16, 1289−1298. (30) Politi, Y.; Metzler, R. A.; Abrecht, M.; Gilbert, B.; Wilt, F. H.; Sagi, I.; Addadi, L.; Weiner, S.; Gilbert, P. U. P. A Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 17362−17366. (31) Petsko, G. A.; Ringe, D., Eds. In Protein Structure and Function: From Sequence to Consequence; Blackwell: Oxford, U.K., 2003; Chapter 1, p 14. (32) Penn, R. L.; Banfield, J. F. Science 1998, 281, 969−971. (33) Gehrke, N.; Cölfen, H.; Pinna, N.; Antonietti, M.; Nassif, N. Cryst. Growth Des. 2005, 5, 1317−1319. (34) Nassif, N.; Pinna, N.; Gehrke, N.; Antonietti, M.; Jäger, C.; Cölfen, H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12653−12655. (35) Addadi, L.; Weiner, S. Angew. Chem., Int. Ed. 1992, 31, 153− 169. (36) Meenakshi, V. R.; Martin, A. W.; Wilbur, K. M. Mar. Biol. 1974, 27, 27−35. (37) Zhang, G. S.; Huang, Z. Q. Opt. Express 2010, 18, 13361− 13367.

growing, fusing, and rotating nanocrystals, and aggregating nanorods in a 2D manner (Figure 8f−h). It is interesting these ANAs are strikingly similar to microstructures of the cephalopod mollusk Nautilus pompilius and Nautilus macromphalus shells.35,36 More interestingly, the ACC sheathed nanorod comprising many oriented attached nanocrystals (Figure 6e) is also strikingly similar to aragonite fibers in natural bivalve ligaments (Figures 5d and 3c in refs 20 and 37, respectively). These similarities and our results indicate that the formation of aragonite fibers in S. radiata ligament is controlled by K58.

5. CONCLUSIONS This article shows K58 alone is sufficient to control the growth of almost pure ANA with unusual morphologies via transformation of ACC at ambient conditions. It should be the short-range order of carboxyl groups of K58 inducing the formation of ACC with short-range order resembling aragonite crystal, resulting in the preferential transformation of ACC into aragonite crystal. Our findings will help for better understanding of the formation mechanism of aragonite fibers in bivalve ligaments and will also contribute some valuable information to the aragonite crystal growth process and to biomineralization researches.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-771-3270152. Fax: +86-771-3270152. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Research Centre for Proteome Analysis, Key Lab of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for LC/MS−MS testing. We also acknowledge National Engineering Research Centre for Domestic Building Ceramics for TEM, HRTEM, and SAED measurements. Thanks also are given to Jiang Minjie for his assistance in FTIR testing and Liu Leping for his valuable discussions. This work was financially supported by the National Natural Science Foundation of China (Grant No. 40772033).

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REFERENCES

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