Selective in Vitro Effect of Peptides on Calcium Carbonate

Crystal Growth & Design , 2002, 2 (5), pp 387–393 ... SEM, FTIR, and confocal microsopy techniques were used to monitor the ... vaterite crystallite...
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Delivered at the Crystal Engineering to Crystal Growth: Design and Function Symposium, ACS 223rd National Meeting, Orlando, Florida, April 7-11, 2002

CRYSTAL GROWTH & DESIGN 2002 VOL. 2, NO. 5 387-393

Selective in Vitro Effect of Peptides on Calcium Carbonate Crystallization Chunmei Li, Gregory D. Botsaris,* and David L. Kaplan Department of Chemical and Biological Engineering & Bioengineering Center, Tufts University, Medford, Massachusetts 02155 Received June 24, 2002;

Revised Manuscript Received July 25, 2002

ABSTRACT: Peptides carried by filamentous phages with potential affinity to calcium carbonate were selected experimentally from a phage display library. The isolated phage were employed to affect the precipitation of calcium carbonate from aqueous solution. SEM, FTIR, and confocal microsopy techniques were used to monitor the morphology and polymorph development of calcium carbonate crystallization. Hollow spheres consisting of nanoscale vaterite crystallites were obtained, which may be due to the template effect of the phage. These spherical crystals transformed into rhombic calcite crystals with time, a process that was slowed by the presence of phage. It is proposed that this phase transformation is a solution-mediated process. Introduction Nature uses organic matrixes to control and direct the crystallization of minerals. This control is exerted through the use of organic macromolecules that provide sites of nucleation and dictate crystal orientation and crystal morphology. As a general model, the organic matrix consists of a structural framework of hydrophobic macromolecules (water insoluble macromolecules: WISM) in association with acidic macromolecules (water soluble macromolecules: WSM) that act as a nucleation surface for biomineralization. An understanding of the mechanisms by which these molecules function can provide new routes to synthesize materials with desired properties (crystal size, morphology, orientation) through molecular-level recognition between the organic and inorganic phase. Numerous in vitro studies have been carried out to mimic biomineralization process at organic-inorganic interfaces. Control of crystallization of inorganic compounds is affected either by an organic template that mimics the lattice of a two-dimensional face of the inorganic phase or by the stereochemistry of the organic functional groups at the interface. One of the most intensively examined systems is calcium carbonate. Model systems have been used to study the possible roles of macromolecules in the control of calcium carbonate crystallization. Small molecules and peptides analogues1,2 have a strong effect on CaCO3 crystallization. Langmuir monolayers,3-11 ultrathin organic films,12 thiol-gold self-assembled monolayers,13-15 and foam lamellae16 have been used as templates for the growth * Corresponding author: Chemical and Biological Engineering Department, Tufts University, 4 Colby St, Medford, MA 02155. E-mail: [email protected]

of CaCO3 crystals, including the control of polymorph and crystal orientation. Substrates such as collagenous matrixes,17-20 polymer substrates,21 and crystal-imprinted polymer surfaces22 have been used to control the nucleation of CaCO3 crystals. A class of functional polymers, the so-called double-hydrophilic block polymers, has been used to control the polymorph and morphologies of CaCO3 crystals.23,24 Normally, WISM provides the mechanical support for biomineralization, while WSM bears the functional groups involved in controlling nucleation. The acidic WSM often contains a significant content of aspartic and glutamic acids, as well as serine and threonine amino acids that are modified with covalently bound phosphate groups. Acidic glycoproteins often contain sulfate and carboxylic acid residues.25 Molecular cloning has provided some information on the repeat sequences present in the WSM of nacrein, a polyanionic macromolecule extracted from the nacreous layer of the Japanese pearl oyster.26 Sequence repeats of [Gly-Xaa-Asn] (Xaa ) Asp, Asn, or Glu) have been identified in nacrein. However, the elucidation of other proteins involved as templating molecules in calcium carbonate mineralization has been difficult. Alternatively, phage display (Phage display describes a selection technique in which a peptide or protein is expressed as a fusion with a coat protein of a bacteriophage, resulting in display of the fused protein on the exterior surface of the phage virion, while the DNA encoding the fusion resides within the virion.29) libraries, based on a combinatorial library of random peptides, allow a large family of different peptides to be screened for affinity against CaCO3 crystals. In this self-selection process, the peptides with amino acid sequence capable of forming functional interactions with calcium carbonate are isolated. For

10.1021/cg0255467 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/04/2002

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Figure 1. CaCO3 crystals obtained in the presence of different phage (a) control, scale bar ) 5 µm, (b) 3R32, scale bar ) 10 µm (c) 4R12, scale bar ) 5 µm, and (d) 5R39, scale bar ) 5 µm. Conditions: [phage] ) 1012/mL, [Ca2+] ) [HCO3-] ) 20 mM, pH ) 8.7, 2-h samples.

example, a 15-mer peptide with a secondary structure that preferentially interacts with calcium carbonate. Since the selected phage pool can be amplified by propagation in Escherichia coli, multiple rounds of selection can be carried out to iteratively select for the strongest binding sequences. A successful application of this technique in inorganic materials is the work of Whaley et al. in which a 12-mer random phage display library was used to bind to a range of semiconductor surfaces with high specificity, depending upon the crystallographic orientation and composition of the structurally similar materials used.27 In the present study, a 15-mer phage display library28 was used for selection of peptides that bound to CaCO3 surfaces. The selected peptides were used to study control of crystallization of CaCO3 in the form of carrier phage. Experimental Section Affinity Selection. A 15-mer peptide library displayed on Fd-tet filamentous phage was donated by G. Smith of the University of Missouri, Columbia.28,29 The calcite crystal was Iceland spar (WARD’S Natural Science Establishment, Rochester, NY). Two columns were prepared from 3 mL syringes, one as a control (no crystals) and the other one packed with freshly cleaved crystals (about 3 × 3 × 3 mm). The library was passed through a column packed with calcite crystals and rocked for 1 h. The crystals were washed with Tris buffered saline (pH 7.5) containing Tween-20. The phage were eluted from the surface of the crystal by the addition of glycine-HCl (pH 2.2) for 30 min, collected in a fresh tube and neutralized with Tris-HCl (pH 9.1). The diluted phages were tittered. Five rounds of affinity selection were carried out. Phages collected after the third round were used to infect E. coli TG1 and plated

on LB-tet plates. Plaques were propagated and DNA sequenced (10 to 20 clones per round) using standard methods. Solution Crystallization. Crystallization was carried out by mixing the calcium containing solution (CaCl2, 20 mM) and carbonate containing solution (NaHCO3, 20 mM) at two different pH values, 8.7 and 10. The pH of the NaHCO3 was adjusted to the required pH with 1 M NaOH. An appropriate amount of amplified phage was included in the carbonate containing solution. The two solutions were mixed by rapid introduction of 750 µL of CaCl2 solution into the equal volume of NaHCO3 solution. After the solution was stirred for 10 min, the suspension was kept in a thermostat (T ) 22 °C) for different periods of reaction time (2 h to 2 months) prior to characterization. Characterization. FTIR (Bruker Equinox55, Billerica, MA) was used for the determination of polymorphic composition of the CaCO3 crystals. Two operation modes were used. The precipitated crystals were collected on a ZnSe FTIR window, washed with deionized water, and dried in a desiccator at room temperature. The window was examined under a FTIR microscope. For larger crystals, the dried crystals were ground for analysis. Scanning electron microscopy (Leo 982 and JEOL JSM840A) was used for morphology assessment of CaCO3 crystals. The crystals were collected on a round cover glass (1.2 cm), washed with deionized water, and dried in a desiccator at room temperature. The cover glass was then mounted on a SEM stub and coated with gold for SEM analysis. Preparation and Imaging of Fluorescent-Labeled Phage. The phage was fluorescently labeled with a dye, YOYO-1 (1 mM in DMSO, Molecular Probes, Eugene, OR), which is fluorescent when bound with phage DNA and nonfluorescent in the unbound state. Five hundred microliters of amplified phage were mixed with 50 µL of YOYO-1 dye, which corresponds to a base pair/dye ratio of 1:5. The mixture was incubated at 4 °C in dark for 72 h. The phage were then

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Figure 2. SEM images illustrating the morphological transformation in the presence of 4R12: (a) 2 h, scale bar ) 5 µm, (b) 24 h, scale bar ) 10 µm, (c) 24 h, scale bar ) 2 µm, (d) 14 days of aging in mother liquid, scale bar ) 20 µm. Conditions: [phage] ) 1012/ml, [Ca2+] ) [HCO3-] ) 20 mM, pH ) 8.7. dialyzed changes Odyssey WI) was

in a 3-mL Slide-A-Lyzer (Pierce) against three of ∼1 L of cold TBS. A confocal microcope (Noran XL laser scanning confocal microscope, Middleton, used to detect labeled phage.

Results and Discussion Phage Sequence Selection. Amino acid composition translation from the sequenced phage selected during affinity selection was completed for each round of selection. The ratio of the observed frequency to the nominal frequency shows the extent of enrichment of each amino acid.30 Except for some hydrophobic amino acid residues (Phe, Ala, Gly), Asp/Glu and Ser/Thr were the most represented residues; affinity selection against calcite resulted in the enrichment of Asp, Glu, Ser, and Thr. These amino acid residues contain hydroxylate (Asp and Glu) or hydroxyl (Ser and Thr) groups in their side chain, that can coordinate with calcium ions on the calcite surface. With Asp, Glu, Ser, and Thr being considered as equivalent residues, a pattern search (TEIRESIAS, Bioinformatics and Pattern Discovery Group, IBM) was performed for sequence data of each round of affinity selection. Patterns, such as [AspGluSerThr]-Xaa-Xaa-[AspGluSerThr], Ser-[AspGluSerThr], Ser-Xaa-Xaa-Ser, were identified. Each peptide sequence was scanned against PROSITE. The phosphorylation site for protein kinase C was found in seven sequences from the affinity selection, 3R1, 3R35, 4R4, 4R6, 4R8, 4R12, and 5R39. (3R9 represents the ninth clone sequenced from the third round of affinity selection.) The conserved pattern was [SerThr]-Xaa-[ArgLys]. The phosphorylation site for casein kinase II was

Table 1. Selected Amino Acid Sequences of Peptides Inserts sequence 3R9 3R11 3R32 3R35 4R12 4R17 4R29 5R4 5R39

amino acid sequence S S A G A R R G G

E F S D Y L G W R

E L V S G S D R V

L A S L S G M N L

L L R H S A S L A

V P S S G H L S G

E A E A F F L M S

S S V D Y L G L S

S H L G S S E A A

A S G A A T F L V

I V V T S S T G S

R M A S F Y S S S

S F Y R T D P D R

R R L F P V Y S P

E G V Y R R G L S

found in 3R14 and 4R17. The conserved pattern of casein kinase II phosphorylation site was [SerThr]Xaa-Xaa-[AspGlu]. These findings support the potential role for phosphorylated Ser or Thr residues in affinity selection process. The Ser and Thr might be in the phosphorylated state and the phosphate group is an effective chelating agent for calcium ions, as is the carboxyl group in the Asp/Glu side chain. This resembles a special class of proteins related to another calcium containing biomineral, hydroxyapatite. These proteins contain multiple phosphoseryl residues and in most cases are found associated with other acidic residues.25 The role of serine and phosphorylated serine in calcium carbonate crystallization will be explored in a subsequent study with synthesized peptides. On the basis of the above analysis, nine clones were selected as potential regulators for CaCO3 crystallization. The sequences of peptide inserts of the selected clones are shown in Table 1. Crystal Morphology Development. Crystallization in the presence of phage was studied at two different

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Figure 3. Hollow spherical structures formed with phage: (a), (b): control, (c), (d): 4R12, scale bar ) 2 µm. Conditions: [phage] ) 1012/mL, [Ca2+] ) [HCO3-] ) 20 mM, pH ) 10.

Figure 4. CaCO3 crystals obtained after aging for 24 h: (a) Control, scale bar ) 20 µm (b) 4R12, scale bar ) 20 µm added at t ) 20 min. Conditions: [phage] ) 1012/mL, [Ca2+] ) [HCO3-] ) 20 mM, pH ) 8.7.

pH values, 8.7 and 10. Figure 1 shows a typical SEM image of CaCO3 crystals obtained (pH 8.7) in the presence of three of the nine phage clones selected. This reaction was run with a phage concentration of 1012/ mL, a CaCO3 concentration of 20 mM, and a starting pH of 8.7. After aging of the samples for 2 h, spherical aggregates of primary particles were obtained. For a control without phage, crystals with a wide size distribution were obtained. With phage, the aggregation state and the size of primary particles differed depending on the phage used. For example, in case of 3R32, not only the aggregation of primary particles, but also coalescence of spherical particles was observed. Crystals obtained in the presence of 4R12 were well dispersed spherical particles with rough surfaces, while in the

presence of 5R39 the particles were spherical with a smooth surface. By monitoring the growth of CaCO3 crystals, morphology development was observed as shown in Figure 2. The spherical particles transformed into rhombic crystals after a longer aging period in mother liquid. For the control without phage, the transformation process was completed within 24 h as shown in Figure 2. With phage, the morphology transformation was delayed until ∼72 h. An interesting feature of the spherical particles was that they adopted a hollow sphere morphology at both low (8.7) and high (10) pH. Colfen and Antonietti 24 also reported the formation of hollow vaterite particles in the presence of double-hydrophilic block polymer CH3O-

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Figure 5. Spherical structure observed with confocal microscopy: (a) 4R12, unlabeled phage (b) 4R12, YOYO-1 labeled phage, pH ) 10, (c) 4R12, YOYO-1 labeled phage, pH ) 8.7, scale bar ) 25 µm. Conditions: [phage] ) 1012/mL, [Ca2+] ) [HCO3-] ) 20 mM.

Figure 6. FTIR spectra of CaCO3: (a) 2 h, (b) 24 h, (c) 48 h, (d) 72 h.

poly (ethylene glycol)-b-poly (ethylenimine)-poly (acetic acid) (PEIPA). They speculated that PEIPA adsorbed on the surface of the amorphous particles of the precipitated CaCO3 and acted as template for nucleation of vaterite which then grew on the outside at the expense of the unstable amorphous particle core leading to a hollow sphere. The spherical vaterite is an aggregate of primary nanoscale vaterite particles which formed in bulk solution through spontaneous precipitation. The phage may form a suprastructure upon which the primary vaterite particles adsorb and grow, resulting in hollow spherical structures. With phage (Figure 3c,d), primary particles formed a shell and a hollow interior revealed. In contrast, without phage (Figure 3a,b), random aggregation of primary particles led to

the formation of solid spherical structures of various sizes (in particular compare the half-spheres in Figure 3b,d). Another observation, which will be discussed later in this paper, showed that the presence of the phage slowed appreciably the transformation of the initial polymorph (vaterite) to calcite, from 24 h in the case of control experiment to 72 h in the case of phage. In an experiment designed to identify the process stage at which phage affect the crystallization of calcium carbonate, phage were introduced 20 min after the onset of the precipitation. The result was that the morphological transformation was completed within 24 h (Figure 4). Therefore, the addition of the phage after the formation of the solid spheres of vaterite had no effect on the rate

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of their transformation to calcite. It can be concluded that phage can act either at the initial of nuclei formationsadsorbing on them and retarding their transformationsor by somehow stabilizing the resulting hollow spherical particles. To further explore the effect of phage on the formation of hollow structures, fluorescently labeled phage was used in calcium carbonate crystallization. Fluorescently labeled phage were visualized under confocal microscopy (Figure 5). The phage organization was similar to that observed for the corresponding spherical crystals, supporting the template effect of phage mentioned earlier. For example, at pH of 10, each spot in Figure 5b (2∼5 µm) corresponds to one spherical structure which is similar to those in Figure 4c,d (also 2∼5 µm). Phase Transformation. Calcium carbonate has three polymorphs: calcite, aragonite, and vaterite. Calcite is the thermodynamically most stable form and vaterite is the least stable.28 FTIR was used to determine the polymorph of calcium carbonate obtained in the presence of the phages. This method has been used successfully for polymorph determination.31,32 The spherical particles formed at the early period of the experiment were vaterite (Figure 6a). The characteristic adsorption peaks of vaterite are at 877 and 744 cm-1. This is consistent with the Ostwald-Lussac rule of stages,33 which states that the initial mineral formed from a solution supersaturated with respect to more than one mineral is the one with the highest solubility, that is, vaterite. The spherical hollow vaterite crystals transformed into calcite with time (Figure 6b-d). The characteristic peaks of calcite are at 876 and 713 cm-1.31,32 There are two possible transformation paths from the unstable to stable polymorph34: (i) solid-state transition and (ii) solution-mediated transformation. In the first case, the internal rearrangement of the crystal lattice occurs. In the second, the transformation takes place through two simultaneous processes, the dissolution of the unstable phase and the nucleation and growth of the stable phase. SEM studies showed that two kinds of rhombic crystals formed, one with smooth surfaces and the other with holes on the crystal surface. The calcite nuclei may form either in solution or in spaces between spherical crystals. Crystals with smooth surfaces come from the solution nuclei. The holes are apparently caused by the hollow spherical vaterite particles, which suggested that rhombic crystals evolved from the calcite nuclei formed between vaterite aggregations. The 2-h sample (4R12, pH ) 8.7, data not shown) was aged in air at room temperature. FTIR spectra at different time points showed that no phase transformation occurred over a two-month period, which implied that solid-state transformation was not occurring. Therefore, one could conclude that the solution-mediated phase transformation occurred through the dissolution of vaterite and crystallization of calcite. This agrees with previous phase transformation studies from vaterite to calcite studies.35-39 Compared with control experiments without phage, the phase transformation process was slower in the presence of phage, which suggests that the phage stabilize vaterite and/or inhibit the growth of calcite. At pH ) 8.7, the transformation was delayed from 24 h

Li et al.

(without phage) to 72 h (with phage). Delay of transformation from vaterite to calcite was also reported in the presence of simple ions, such as Li,35 Cu 2+,36 and Mg2+,39 and the double-hydrophilic block polymer 25. The vaterite can be stabilized for about one year in the case of the hydrophobic block polymer. Conclusion Phage selected by their adsorption on CaCO3 crystals from a phage display library were used to study the crystallization of CaCO3. Hollow spheres consisting of primary nanoscale particles were obtained due to the template effect of the phage. The phase transformation process was accompanied by a morphological transformation process. Phage slowed the transformation process from vaterite to calcite. The data point to the transformation from vaterite to calcite being a solutionmediated process. The findings have implications for phage/peptide-mediated control of crystallization processes. In the continuation of this study, synthesized peptides of the selected sequences will be used. Acknowledgment. We thank the NSF for the support of this work through Grant CTS-9986545 and the Keck Foundation. References (1) Mann, S.; Didymus, J. M.; Sanderson, N. P.; Heywood, B. R.; Samper, E. J. A. J. Chem. Soc., Faraday Trans. 1990, 86, 1873-1880. (2) Gower, L. A.; Tirrell, D. A. J. Cryst. Growth 1998, 153160. (3) Mann, S., Heywood, B. R., Rajan, S., Birchall, J. D. Nature 1988, 334, 692-695. (4) Didymus, J. M.; Mann, S.; Benton, W. J.; Collins, I. R. Langmuir 1995, 11, 3130-3135. (5) Ahn, D. J.; Berman, A.; Charych, D. J. Phys. Chem. A 1996, 100, 12455-12461. (6) Lahiri, J.; Xu, G.; Dabbs, D. M.; Yao, N.; Aksay, I. A.; Groves, J. T. J. Am. Chem. Soc. 1997, 119, 5449-5450. (7) Rajan, S.; Heywood, B. R.; Walker, J. B. A.; Mann, S J. Chem. Soc., Faraday Trans. 1991, 87, 727-734. (8) Heywood, B. R.; Rajan, S.; Mann, S. J. Chem. Soc., Faraday Trans. 1991, 87, 735-743. (9) Heywood, B. R.; Mann, S. Chem. Mater. 1994, 6, 311-318. (10) Litvin, A. L.; Samuelson, L. A.; Charych, D. H.; Spevak, W.; Kaplan, D. L. J. Phys. Chem. 1995, 99, 12065-12068. (11) Litvin, A. L.; Suresh V.; Kaplan, D. L. Mann S. Adv. Mater. 1997, 2, 124-127. (12) Archibald, D. D.; Qadri, S. B.; Gaber, B. P. Langmuir 1996, 12, 538-546. (13) Kuther, J.; Nelles, G.; Seshadri, R.; Schaub M.; Butt, H. J.; Tremel, W. Chem.-Eur. J. 1998, 4, 1834-1842. (14) Aizenberg, J.; Black, A. J.; Whitesids, G. M. Nature 1999, 398, 495-498. (15) Aizenberg, J.; Black, A. J.; Whiteside, G. M. J. Am. Chem. Soc. 1999, 121, 4500-4509. (16) Chen B. D.; Cilliers, J. J.; Davey, R. J.; Garside, J.; Woodburn, E. T. J. Am. Chem. Soc. 1998, 120, 1625-1626. (17) Falini, G.; Fermani, S.; Gazzano, M.; Ripamonti, A. Chem.Eur. J. 1997, 3, 1807-1812. (18) Falini, G.; Fermani, S.; Gazzano, M.; Ripamonti, A. Chem.Eur. J. 1998, 4, 1048-1052. (19) Falini, G. Int. J. Inorg. Mater. 2000, 2, 455-461. (20) Falini, G.; Fermani, S.; Gazzano, M.; Ripamonti, A. J. Chem. Soc., Dalton Trans. 2000, 3983-3987. (21) Dalas, E.; Klepetsanis, P.; Koutsoukos, P. G. Langmuir 1999, 15, 8322-8327. (22) D’Souza, S. M.; Alexander C.; Carr, S. W.; Waller, A. M.; Whitcombe M. J.; Vulfson E. N. Nature 1999, 398, 312316.

Effect of Peptides on Calcium Carbonate Crystallization (23) Colfen H.; Qi, L. Chem.-Eur. J. 2001, 7, 106-116. (24) Colfen H.; Antonietti, M. Langmuir 1998, 14, 582-589. (25) Mann, S. in Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: New York, 2001. (26) Miyamoto, H.; Miyashita, T.; Okushima, M.; Nakano, S.; Morita, T.; Matsushiro, A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 9657-9660. (27) Whaley, S. R.; English, D. S.; Hu E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665-668. (28) Scott, J. K.; Smith, G. P. Science, 1990, 249, 386-390. (29) Smith, G. P. http://www.biosci.missouri.edu/smithgp/ index.html: Phage-Display Vectors and Libraries Based on Filamentous Phage Strain fd-tet. (30) Instruction Manual, Ph.D.-12TM Phage Display Peptide Library Kit, New England Biolabs. (31) Lippmann F. in Sedimentary Carbonate Minerals; Springer Verlag: Berlin, 1973.

Crystal Growth & Design, Vol. 2, No. 5, 2002 393 (32) Falini, G.; Albeck, S.; Weiner S.; Addadi, L. Science 1996, 271, 67-69. (33) White W. B. in Infrared Spectra of Minerals; Mineralogical Society: London, 1974. (34) Davey, R. J.; Cardew, P. T.; McEwan D.; Sadler, D. E. J. Cryst. Growth 1986, 79, 648-653. (35) Ogino, T.; Suzuki, T.; Sawada, K. J. Cryst. Growth 1990, 100, 159-167. (36) Nassrallah-Aboukais, N.; Boughriet, A.; Laureyns, J.; Aboukais, A.; Fischer, J. C.; Langelin, H. R.; Wartel, M. Chem. Mater. 1998, 10, 238-243. (37) Spanos, N.; Koutsoukos, G. J. Cryst. Growth 1998, 191, 783-790. (38) Kralj, D.; Brecevic, L.; Kontrec J. J. Cryst. Growth 1997, 177, 248-257. (39) Kitamura, M. J. Colloid Interface Sci. 2001, 236, 318-327.

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