Nacre Protein Sequence Compartmentalizes Mineral Polymorphs in

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Nacre Protein Sequence Compartmentalizes Mineral Polymorphs in Solution Jong Seto,*,†,‡ Andreas Picker,‡ Yong Chen,† Ashit Rao,‡ John Spencer Evans,§ and Helmut Cölfen‡ Department of Chemistry, École Normale Supérieure, Laboratoire P.A.S.T.E.U.R. (UMR 8640 CNRS-ENS-UPMC), 24 rue Lhomond, 75005 Paris, France ‡ Department of Chemistry, Physical Chemistry, Universität Konstanz, Universitätstrasse 10, Konstanz D-78457, Germany § Division of Basic Sciences and Craniofacial Biology, New York University, New York, NY 10010, United States †

S Supporting Information *

ABSTRACT: The Japanese pearl oyster (Pinctada fucata) n16 framework matrix protein is an integral part of the growth and formation of the mollusk shell biomineralization mechanism. It is a required component of the extracellular matrix with a dual mineralization role, as an anchor component to synchronize the assembly of the beta-chitin and N-series, Pif-series protein extracellular matrix for aragonite formation and as a regulator of aragonite formation itself. However, the mechanism by which this protein controls aragonite formation is not understood. Here, we investigate the mineralization potential and kinetics of the 30 AA N-terminal portion of the n16 protein, n16N. This sequence has been demonstrated to form either vaterite or aragonite depending upon conditions. Using in situ potentiometric titration methods, we find that n16N is indeed responsible for the self-assembly characteristics found in vivo and in vitro but is not involved with active Ca2+ binding or mineral nucleation processes. Upon the basis of time- and peptide concentration-dependent sampling of mineral deposits that form in solution, we find that n16N is responsible for controlling where mineralization occurs in bulk solution. This protein sequence acts as a molecular spacer that organizes the mineralization space and promotes the formation of mineral constituents that contain ACC, vaterite, and aragonite. Without the concerted action of the n16N assemblage, unregulated calcite formation occurs exclusively. Thus, the n16 protein provides the regulation needed to have the characteristic polymorph, crystalline orientations, and related mechanical properties associated to the microstructure of mollusk shells.

T

lattice structures).13−16 In the mollusk shell, polymorph selection is under the control of specific aggregation-prone extracellular matrix protein assemblies found in each shell layer.1−3,12,17−19 In the nacre layer, two classes of proteins form assemblies: the intracrystalline series, which are occluded within the mature aragonite single crystals,10,12,17−19 and the framework series that associates with the beta chitin polysaccharide layer of aragonite tablets.1−3 Unfortunately, the mechanisms utilized by both nacre protein families to stabilize aragonite are unknown. To address this shortcoming, we initiated bulk solution pH-controlled (constant at pH 9.75) potentiometric titration studies of a wellcharacterized nacre framework protein sequence, n16N.4−6 This 30 AA N-terminal sequence oligomerizes to form supramolecular assemblies (films and particles) that, like the parent protein n16,1−3 promote the nucleation of vaterite or aragonite, depending upon conditions. Hence, it represents an ideal model system for establishing how nacre protein sequences affect the nucleation process, prenucleation cluster formation in solution, and polymorph transformation.20

he Japanese pearl oyster (Pinctada fucata) n16 framework matrix protein is an integral part of the oyster shell aragonite nacre layer.1−3 This sequence has been demonstrated to form either vaterite or aragonite in vitro, depending upon solution conditions.1−3 However, the mechanism by which this protein controls polymorph formation is not understood. Here, we investigate the effect of the 30 AA N-terminal region of the n16 protein, n16N,4−6 on calcium carbonate mineralization using in situ potentiometric titration methods. On the basis of time and peptide concentration-dependent sampling of mineral deposits that form in vitro, we find that n16N is responsible for controlling where mineralization occurs in bulk solution. This protein sequence self-assembles to create small-volume compartments that arrange and stabilize vaterite and aragonite biominerals without transitioning through ACC. In the absence of n16N, unregulated calcite formation occurs exclusively. Thus, this nacre protein sequence has the inherent capability of both polymorph stabilization and spatial mineral organization, key traits that are important for the material properties of mollusk shells.7−11 The formation of opposing layers of single crystal calcite and aragonite in the mollusk shell7−11 represent biogenic polymorphism (i.e., identical chemical compounds with different © 2014 American Chemical Society

Received: September 23, 2013 Revised: March 4, 2014 Published: March 7, 2014 1501

dx.doi.org/10.1021/cg401421h | Cryst. Growth Des. 2014, 14, 1501−1505

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intrinsically disordered n16N sequence will interact with Ca2+ and other metal ions but does not fold.30 This suggests that n16N binding affinity to Ca2+ may be weak. To verify this, we extended our potentiometric titration measurements of n16N as a function of pH to examine n16N-Ca2+ binding affinity under mineralization conditions. Here, it was found that n16N does not display any binding interaction with Ca2+ ions. In situ potentiometric measurements at pH = 7.4, 8.0, 9.75, and 12 revealed no significant levels of n16N−Ca2+ binding activity, rather than effects from ionic strength due to pH variation. However, note that in Figure 1, a lower amount of Ca2+ ion is present in solution during nucleation compared to the reference case, indicating a specific amount of Ca2+ ions sequestered from the bulk solution. This indicates that these Ca2+ ions are bound in prenucleation clusters rather than to n16N. A possible mechanism that can lead to sequesteration of these clusters and subsequently, Ca2+ ions, can be attributed to direct interactions of the bound carbonate and/or water moieties by n16N.31−33 Combining these two present observations with the results obtained from previous studies,30 we conclude that although n16N is not a strong Ca2+ ion binder, it can effectively sequester Ca2+ from the solution by changing the prenucleation cluster equilibrium, increasing the amount of Ca2+ ion bound in the clusters and concentrate Ca2+ ions by a novel structuring scheme. We next examine the effect n16N has on the equilibrium concentration after nucleation, which corresponds to the solubility product (Figure S1 of the Supporting Information). Typically, the solubility products are expected to be in the range of 3.1 × 10−8 (ACC-I) or 3.8 × 10−8 (ACC-II) after nucleation (pH 9.75, 25 °C).20,23 In the presence of low n16N concentrations, all solubility products are found to be approximately 3.8 × 10−8 M2, which corresponds closely to ACC-II, which is the expected product at pH 9.75 and is a precursor of vaterite (Figure S1 of the Supporting Information).20 However, at higher n16N concentrations (370 μg/mL) (Figure S1 of the Supporting Information), we find that the solubility product after nucleation is 1.3 × 10−8 M2, which closely corresponds to vaterite (1.22 × 10−8 M2).20 Thus, above a specified concentration range a nacre protein sequence promotes vaterite nucleation directly from prenucleation clusters without detectable transitioning steps through ACC. Analysis of the mineral morphologies formed after the nucleation and ripening processes reveals that n16N acts as a polymorph selector. Characteristic rhombohedral calcite crystals are predominantly observed in both the control and the 0.1 μg/ mL (i.e., 27 nM) n16N scenarios (Figure S7 of the Supporting Information). However, in the presence of n16N, we note that the calcite crystals are clustered together, a phenomenon not observed in the negative controls. At 1 μg/mL (270 nM) n16N, the titrants can be observed to possess more calcite crystals along with a competing fraction having a spherical morphology (Figure 2 and Figure S7 of the Supporting Information). These spheres nonpreferentially aggregate and this spherical fraction is from ∼500 nm to 2 μm in diameter. Electron diffraction patterns (Figure 2 and Figure S7 of the Supporting Information) as well as polarized light microscopy, XRD, and FTIR analyses (Figures S2, S3, S5 and Table S1 of the Supporting Information) confirm that this spherical mineral phase is vaterite. This finding coincides with both the observed vaterite-specific solubility product (Figure S1 of the Supporting Information). Stabilizing vaterite may not be an uncommon phenomena in mollusc mineralization by small proteins,34 but n16N is able to organize these mineral fractions such that subsequent crystal growth and transformation

Our initial observation is that at low n16N concentrations, there is no observable effect on the nucleation process itself, as evidenced by no significant differences over the time course of Ca2+ ion addition in comparison to negative controls (with the exception of the slope prior to nucleation) (Figure 1). Although

Figure 1. In situ potentiometric titration of 10 mM CaCl2 into 10 mM Na2CO3 buffer in the presence and absence of n16N, pH 9.75. The inset graph is an expansion of earlier time points. Reference refers to no peptide added. The slope of each titration curve is proportional to the amount of formed CaCO3 prenucleation clusters20 with a smaller slope indicating a larger number of cluster bound Ca 2+ ions. We observe a significant influence of n16N onto the prenucleation cluster equilibrium with the exception of the curve for 10 μg/mL, which is reproducibly found to be very similar to the reference curve for a yet unclear reason. The amount of bound Ca2+ ions is more than double for the 100 μg/mL sample as compared to the reference case without additives.

Table 1. Kinetics of Nucleation and Ripening sample

tnucla

tripea

tnucl/ripea

Δtnucl, ripe/tripe (%)

control 0.1 μg/mL 1 μg/mL 10 μg/mL 100 μg/mL

43 46.1 47.5 38.5 47.3

51 61.5 61.5 55 67.3

8 15.4 14 16.5 20

15.7 25 22.8 30 29.7

a

Units are in minutes. Abbreviations: Nucl = nucleation and ripe = ripening.

we do observe that the time intervals when nucleation and ripening occur are slightly altered by n16N relative to the control scenario (Table 1), these changes are minor compared to the reported nucleation inhibition observed for synthetic polyelectrolyte additives.21,22 Thus, n16N does not significantly accelerate or delay the nucleation and ripening processes and has little effect on the induction time. However, with increasing n16N concentration, the slope prior to nucleation is continuously decreasing (Figure 1 and Figure S1 of the Supporting Information ). This shows that the amount of prenucleation clusters is increasing with the n16N concentration; or in other words, n16N stabilizes CaCO3 prenucleation clusters. From circular dichroism spectroscopy measurements of n16N alone in varying pH environments, n16N is found to shift from more beta strand to alpha helical structures with increasing pH. This suggests that an n16N moiety may be involved modulating mineralization-related activities as a function of pH (Figure S4 and Table S2 of the Supporting Information). We next examined the interaction of n16N with Ca2+ ions at different pH values above and below the n16N isoelectric point of pH 8.66 as calculated from the amino acid sequence. It is known that 1502



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Figure 2. CaCO3 mineralization in the presence of n16N as analyzed by backscatter SEM (scalebar = 10 μm), secondary SEM (scalebar = 2 μm), TEM (scalebar = 500 nm), and electron diffraction (Electron Diffraction) (0.5 Å−1). Control = no peptide added. In the electron diffraction patterns, letters indicate the polymorph either as calcite (C), aragonite (Aa), and vaterite (V) with the corresponding Miller indices indicated in the diffraction patterns. Scale bars for backscatter SEM, secondary SEM, TEM, and electron diffraction images are 10 μm, 2 μm, 500 nm, and 0.5 Å−1.

talization scheme is clearly noted at 100 μM n16N, where polypeptide film aggregate networks (Figure 3) are observed. These peptide networks contain spaced spherical-to-angular electron dense deposits. Electron diffraction measurements confirm the absence of single crystal diffraction spots, and hence these intrafilm mineral deposits are not calcite or aragonite. The fact that we observe weak intensity diffraction rings (Figures 2 and 3) suggests that these electron-dense particles are either ACC or a poorly crystalline form of vaterite (see Figures S2, S3, and S7 of the Supporting Information), neither of which can be unambiguously identified at this time. Nonetheless, these deposits are clearly organized at periodic intervals within the polypeptide film. Note well that we do not observe the formation of mineral deposits outside of these films. Small-volume compartmentalization is a hallmark of artificial26,27 and natural10,19,28 biomineralizing systems, and thus the ability of the n16N protein sequence to create micrometer-sized compartmentalized mineral deposits is a significant finding. We also observe the same characteristic of assembly and compartmentalization in the full length n16 protein as well (Figure S6 of the Supporting Information). On the basis of our studies, we propose a mechanism by which the n16N nacre protein sequence participates in prenucleation control of calcium carbonate mineralization by way of organization of mineral precursors (Scheme 1). Although prenucleation clusters form independently of n16N participation, this peptide does increase their concentration by stabilization/cluster coordination (Figure 1). Due to the presence of intrinsic disorder and amyloid-like motifs,29 n16N assembles to form films or deposits that create small-volume peptide compartments around mineral clusters (Figure 3). We believe that this compartmentalized mineral

results in hierarchical structures. As shown in Figure 2, spherical particle formation increases as a function of n16N concentration, thus indicating that this fraction forms in direct response to n16N. The appearance of vaterite is important in that it represents a precursor or intermediate phase to aragonite.20−22,24,25 Coincidentally, we observed a twinned crystalline component that localizes on the outer periphery of the spherical clusters (Figure 2 and Figure S7 of the Supporting Information at 1 and 10 μg/mL n16N). This twinned crystalline component strongly resembles aragonite, and we confirmed the presence of this polymorph via electron diffraction (Figure 2) and powder X-ray diffraction (XRD) and Fourier transform-infrared (FT-IR) analyses of bulk deposits (Figures S2 and S3 of the Supporting Information). Note that the presence of aragonite with vaterite is not evident in the solubility product after nucleation (Figure S1 of the Supporting Information) because the solubility of aragonite is lower than that of vaterite and only the most soluble species is detected.20,23 Nonetheless, our experiments establish that n16N is a promotor of aragonite and vaterite polymorphs after nucleation. Interestingly, we only observe the occurrence of polymorphs in clustered assemblies but never as individual deposits. This organizational capability is clearly linked to the presence of n16N (Figures 2 and 3) and takes on interesting characteristics at higher peptide concentrations. At n16N concentrations ≥ 1 μg/ mL, mineral deposits are observed to organize into larger spherical aggregates that orient in a linear fashion. At 100 μg/mL, these aggregates cluster into micrometer-sized “compartments” where the mineralization of a nonspherical fraction is occurring. The direct participation of n16N in this mineral compartmen1503



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Figure 3. Unstained TEM micrographs (at low, intermediate, and high magnification) of n16N films containing mineral deposits. Electron diffraction patterns were obtained for the regions indicated by the arrows (electron dense mineral deposit versus electrolucent peptide film). The corresponding diffraction scalebars = 0.221 Å.

aragonite.4−6 The compartmentalization of mineral precursors then leads to the formation of a vaterite phase via a mechanism whereby vateritic prenucleation clusters are stabilized by n16N before nucleation, and thus, a pathway to a vateritic ACC phase is selected for (Figure 1) after nucleation. The subsequent vaterite mineral phase is observed to be the first crystalline mineral phase laid down and represents an intermediate phase from which the nucleation and growth of an aragonite phase proceeds (Figure 2). From this scheme, a hypothetical deposition of n16N film/ polymorph composite can be conceptualized (Scheme 1) to be the basis for assembly processes of the aragonitic structures reported in earlier in vitro studies2,4 and observed in mollusk shells.9−12,34 For the first time, we demonstrate another mechanism for protein mediation of mineralization: crafting the mineralization space and partitioning the local chemical environments of this space. The n16N peptide, which represents the key selfassembly/aragonite forming domain of the framework protein, n16, achieves control of the mineralization process via two key routes. First, the sequence spontaneously self-assembles to form polypeptide deposits or films that create the mineralization space (Figure 3 and Scheme 1). Second, this sequence commands the spatial sequestration of Ca2+ ions (Figures 1−3), as opposed to direct high affinity Ca 2+ binding or mineral inhibition mechanisms. We believe that this process is key to the formation of the vaterite intermediate and ultimately the aragonite polymorph formation process. Combining these two attributes, this framework protein domain controls the mineralization process in a unique way that has not been documented in the literature to this point. This route for polymorph formation may be representative of the mechanisms employed by framework

Scheme 1. Hypothetical n16N-Mediated Prenucleation Cluster Assembly and 3D Organization Processa

a

Localized compartments are formed by n16N self-assembled organic scaffolds, and these create specialized microenvironments that are conducive to CaCO3 polymorph stabilization. These organic− inorganic assemblies can then extend to form larger supramolecular complexes. (Note: Contrast of n16N films is a result of mineral ions only.)

phase is being stabilized by the peptide assembly, and this hypothesis is supported by earlier n16N studies which noted the ability of these peptide films to stabilize ACC, vaterite, and 1504



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(11) Smith, B. L.; Schaffer, T. E.; Viani, M.; Thompson, J. B.; Frederick, N. A.; Kindt, J.; Belcher, A.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Nature 1999, 399, 761−763. (12) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67−69. (13) Wang, X.; Li, Y. J. Am. Chem. Soc. 2002, 124, 2880−2881. (14) Coa, H. Adv. Mater. 2001, 13, 1393−1394. (15) Desgranges, C.; Delhommelle, J. J. Am. Chem. Soc. 2006, 128, 15104−15105. (16) Torbeev, V. Y.; Shavit, E.; We, I.; Leiserowitz, L.; Lahav, M. Cryst. Growth Des. 2005, 5, 2190−2196. (17) Michenfelder, M.; Fu, G.; Lawrence, C.; Weaver, J. C.; Wustman, B. A.; Taranto, L.; Evans, J. S.; Morse, D. E. Biopolymers 2003, 70, 522− 533. Amos, F. F.; Evans, J. S. Biochemistry 2009, 48, 1332−1339. Amos, F. F.; Ndao, M.; Ponce, C. B.; Evans, J. S. Biochemistry 2011, 50, 8880− 8887. (18) Mann, K.; Siedler, F.; Treccani, L.; Heinemann, F.; Fritz, M. Biophys. J. 2007, 93, 1246−1252. Treccani, L.; Mann, K.; Heinemann, F.; Fritz, M. Biophys. J. 2006, 91, 2601−2608. (19) Fu, G.; Qiu, S. R.; Orme, C. A.; Morse, D. E.; DeYoreo, J. J. Adv. Mater. 2007, 17, 2678−2683. (20) Gebauer, D.; Voelkel, A.; Cölfen H, H. Science 2008, 322, 1819− 1822. (21) Verch, A.; Gebauer, D.; Antonietti, M.; Cölfen, H. Phys. Chem. Chem. Phys. 2011, 13, 16811−16820. (22) Gebauer, D.; Cölfen, H.; Verch, A.; Antonietti, M. Adv. Mater. 2008, 21, 435−439. (23) Brecevic, L.; Nielsen, A. E. J. Cryst. Growth 1989, 98, 504−510. (24) Blank, S.; Arnoldi, M.; Khoshnavaz, S.; Trecccani, L.; Kuntz, M.; Mann, K.; Grathwohl, G.; Fritz, M. J. Microsc. 2003, 212, 280−291. (25) Bischoff, J. L. Am. J. Sci. 1968, 266, 80−90. (26) Loste, E.; Park, R.; Warren, J.; Meldrum, F. C. Adv. Func. Mat. 2004, 14, 1211−1220. (27) Stephens, C.; Ladden, S. F.; Meldrum, F. C.; Christenson, H. K. Adv. Func. Mat. 2010, 20, 2108−2115. (28) Kroeger, N. Science 2009, 325, 1351−1352. (29) Evans, J. S. Bioinformatics 2012, 28, 3182−3185. (30) Collino, S.; Evans, J. S. Biomacromolecules 2008, 9, 1909−1918. (31) Moradian-Oldak, J.; Bouropoulos, N.; Wang, L.; Gharakanian, N. Matrix Biology 2002, 21, 197−205. (32) Osheroff, N.; Brautigan, D. L.; Margoliash, E. Proc. Natl. Acad. Sci. U.S.A. 1980, 77 (8), 4439−4443. (33) Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4410− 4114. (34) Pokroy, B.; Zolotoyabko, E.; Adir, N. Biomacromolecules 2006, 7 (2), 550−556.

nacre proteins, such as Pif-series and the n16 family, to synthesize the nanostructured multilayered aragonite nacre of the shell.

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ASSOCIATED CONTENT

* Supporting Information S

Experimental methods, materials, additional experiments and results, Tables S1-S2, and Figures S1-S7. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +33 (0) 1-4432-2431. Fax: +33 (0) 1-4432-2402. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

Portions of this research were supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award DE-FG02−03ER46099 and represents contribution number 63 from the Laboratory for Chemical Physics, New York University. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Mr. Ashit Y. Rao is thanked for his technical assistance and support. J.S. acknowledges financial support for this work from the Fondation Pierre-Gilles de Gennes in Paris, France. Ms. Reena Bajwa is thanked for her infinite curiosity into nature’s materials and her unrelenting support of this work.

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ABBREVIATIONS n16N = 1−30 AA N-terminal domain of the Pinctada fucata Japanese pearl oyster nacre protein, n16; IDP = intrinsically disordered protein; UDDW = unbuffered deionized distilled water; ACC = amorphous calcium carbonate

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REFERENCES

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Nacre Protein Sequence Compartmentalizes Mineral Polymorphs in

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