Influence of Selected Artificial Peptides on Calcium Carbonate Precipitation - A Quantitative Study Denis Gebauer, Andreas Verch, Hans G. Bo¨rner, and Helmut Co¨lfen* Colloid Chemistry, Max Planck Institute of Colloids and Interfaces, Research Campus Golm, Am Mu¨hlenberg, D-14476 Potsdam-Golm, Germany
CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 5 2398–2403
ReceiVed NoVember 25, 2008; ReVised Manuscript ReceiVed January 28, 2009
ABSTRACT: Calcium carbonate is an abundant biomineral with fascinating shapes and properties. Much effort is spent to study how creatures can control mineral formation. We present a quantitative study of the early stage of calcium carbonate precipitation in the presence of artificial peptide additives, the sequences of which were derived in phage assays to have aragonite binding affinity. A novel crystallization assay shows that the peptide additives inhibit nucleation of calcite. Analysis of the precipitated particles and comparison with nucleation inhibition confirm our recent findings, which suggest that calcitic and vateritic short-range order is already preformed in stable prenucleation clusters, which form amorphous intermediates after nucleation reflecting similar structures and finally become crystalline. In the long run, this process facilitates the control of polymorph formation by the design of the binding affinity of additives to different polymorphs (i.e., the polymorph bound weakest by the additive is to be formed as its formation is least inhibited). These findings facilitate a novel understanding of mineralization control and provide a basis for the analysis of biological peptide sequences and for the analysis of their role in biomineralization processes. Introduction A central question in the study of biomineralization processes is how proteins and other biological macromolecules facilitate control and regulation of nucleation and crystal growth in vivo.1-4 Because of the complexity of biological systems, however, in vivo analyses of control and regulation processes are fairly difficult and are thus simplified by studying in vitro systems. In these, the action of additives such as proteins, polypeptides, low molecular weight compounds, and also ions on mineral formation can be investigated. For example, impressing in vitro experiments revealed that proteins are responsible for the polymorph control of CaCO3 in mollusk shells.5,6 The analysis of the action of additives utilizing in vitro crystallization assays is a fast-growing scientific field providing an almost unmanageable amount of literature (partly summarized in recent review articles7-9). For instance, different proteins associated with the aragonitic nacre layer10-15 and calcitic prismatic layer16-18 of molluscan CaCO3 shells have been identified, and the action of subdomains has been investigated in vitro.19,20 Some of the investigated nacre-associated polypeptides and proteins can act in an orthogonal manner blocking and accelerating different parts of crystal growth,11,21 which is often observed by AFM experiments analyzing CaCO3 growth in the presence of these additives.10,22 Much effort is spent to relate primary and secondary structural features of the polypeptides to the mode of additive action.23,24 In the long run, additives have to interact with a component of the mineral phase to some extent (i.e., a fundamental requirement is additive affinity to a mineral component (ions, clusters, amorphous precursors, crystallites) along the crystallization process). In this study, we chose three artificial model peptides, the sequences of which were deduced in phage assays to have undefined binding affinity to geological aragonite crystals.25 The 12-mer sequences given in the one-character code of amino acids are IHIKFKQHQNHN (peptide 1), KRSKFPHKHDVI (peptide 2), and AVGSTKHKWPPL (peptide 3). In contrast to the acidic biological peptides, which are considered to play important roles in biominerali* To whom correspondence should be addressed. E-mail: coelfen@ mpikg.mpg.de.
zation,19,26 these peptides are rather uncharged and provide basic side chains. However, the binding affinity to geological aragonite is a sufficient criterion for a possible action of the peptides on CaCO3 crystallization. CaCO3 crystallization in the presence of additives is usually performed in Kitano assays27 or utilizing the gas diffusion method,28 which is sensitive toward the crystallization chamber design and the applied crystallization procedure and is thus likely to produce artifacts.29 Furthermore, these crystallization assays can only give qualitative evidence of additive action by means of effects on crystal morphology, polymorphism, or crystallization mechanism and thus cannot show at which stage of crystallization the additives act, unless difficult time-dependent measurements are performed. A quantitative aspect is given by the studies of crystal growth by means of AFM.20 Recently, we reported the results of a novel quantitative CaCO3 crystallization assay, which allows for the detection of all species present in undersaturated and supersaturated solution as well as during the early crystal growth stage.30,31 Dosing of dilute calcium chloride solution into dilute carbonate buffer with simultaneous constant pH titration and recording of the Ca2+ potential facilitates the detection and thermodynamic characterization of stable CaCO3 clusters, which form in the prenucleation stage.30 It could be shown that these stable clusters are the nucleation-relevant species and that pH-dependent clusterformation thermodynamics provides a proximate basis of the formation of presumably structurally different amorphous precursor species, which transform into different polymorphs. Beyond, the novel assay facilitates the categorization of the action of additives during crystallization:31 (I) binding of calcium ions, (II) influence on cluster formation equilibria, (III) inhibition of nucleation, (IV) adsorption of nucleated particles and their stabilization, and (V) influence on the local structure of nucleated particles (i.e., type of amorphous phase or crystalline polymorph). Here, we present the complete quantitative characterization of the action of the three selected peptides, and the analysis shows that the binding affinity of the peptides, though differently pronounced, already acts on clusters forming in the prenucleation
10.1021/cg801292p CCC: $40.75 2009 American Chemical Society Published on Web 03/10/2009
Influence of Peptides on CaCO3 Precipitation
stage as well as on amorphous intermediates, finally influencing the CaCO3 polymorph formed. Experimental Section Peptide Synthesis and Purification. Diisopropylethylamine (DIPEA; IRIS Biotech GmbH, peptide grade), piperidine (Acros, peptide grade), and trifluoroacetic acid (TFA; Acros, peptide grade) were applied as received. Fmoc-amino acid derivatives, N-methyl-2-pyrrolidone (NMP, 99.9+%, peptide synthesis grade), and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were used as received from IRIS Biotech GmbH. 1,3-Dicyclohexylcarbodiimide (DCC, 1 M in NMP), diethyl ether, and triethylsilane were applied as received from Aldrich. Dichloromethane (DCM; IRIS Biotech GmbH, peptide grade) was distilled from CaH2. Wang resin was purchased from Rapp, Polymere GmbH. The loading of the Wang resin (0.24 mmol/g; 0.1 mmol scale) was performed using standard solid-phase-supported peptide synthesis benchtop procedures, applying DCC/DIPEA/NMP protocols.32,33 The oligopeptide synthesis was performed on an Applied Biosystems ABI 433a peptide synthesizer in NMP as solvent following standard ABIFastmoc protocols (double coupling). Amino acid coupling was facilitated using HBTU/DIPEA. The liberation of the different peptides was accomplished by 2-h treatment with the cleavage mixture (95% TFA, 1% triethylsilane, 4% H2O), followed by two washing cycles with DCM. The oligopeptides were isolated by diethyl ether precipitation, centrifugation, and ultrafiltration (MWCO ≈ 1000 g/mol) followed by lyophilization from water. The following 12-mer sequences were prepared: IHIKFKQHQNHN (peptide 1), KRSKFPHKHDVI (peptide 2), and AVGSTKHKWPPL (peptide 3). The chemical integrity of the peptides was characterized by means of ESI-MS in methanol (peptides 1 and 2) or water (peptide 3): Peptide 1: (Mth ) 1543.93 Da): 1544.9 [M + H]1+; 772.0 [M + 2H]2+; 590.3 [M + K]3+ + Na + K; 515.4 [M + 3H]3+ Peptide 2: (Mth ) 1491.92 Da): 796.4 [M + Na]2+ + K; 747.0 [M + 2H]2+; 498.1 [M + 3H]3+ Peptide 3: (Mth ) 1320.73 Da): 1321.7 [M + H]1+; 661.2 [M + 2H]2+; 441.0 [M + 3H]3+ Qualitative in Vitro Crystallization Assay. Five milliliters of 10 mM calcium chloride solution (containing 1 mmol/L peptide 1 (1.544 g), peptide 2 (1.492 g), peptide 3 (1.321 g)) was poured into 20-mL disposable glass, which was covered by Parafilm furnished with three pinholes. The glass was placed together with a disposable glass containing 2 g of ammonium carbonate (also covered with Parafilm furnished with three pinholes) in a desiccator. Coverslips were put on the bottom of the vessel to increase the yield of precipitated crystals. Crystals were yielded after 1 day reaction time. Quantitative in Vitro Crystallization Assay. The utilized commercial Metrohm titration setup and experimental procedure are already described in detail elsewhere.30 In brief, dilute calcium chloride (10 mM) was dosed into dilute carbonate buffer (pH ) 9.00 or 9.75, 10 mM, 25 mL), while the calcium potential was monitored and the pH value was kept constant by means of titration utilizing 10 mM NaOH. The experiment in the absence of additives was the reference experiment. Experiments in the presence of peptide additives were carried out in the same carbonate buffer containing 0.1 g/L peptide. Here, a peptide stock solution (10 g/L) was preset to the particular pH value of the carbonate buffer (pH ) 9.00/9.75), and according to the volume dilution by pH presetting, approximately 1 mL of the pH-set stock solution was filled to 100 mL with the particular carbonate buffer. Thus, dilution of the carbonate buffer was negligible (volume dilution approximately 1%). Circular Dichroism Measurements. Circular dichroism (CD) measurements were performed on a Jasco CD spectrometer (model 715) at 25 °C. Spectra were recorded from 190 to 250 nm at a scanning speed of 50 nm/min with a step length of 0.2 nm. The secondary structures of the peptides were determined by CDSSTR calculations on a base of 48 reference proteins.34-38 Peptide-containing samples were prepared in the following way: A peptide stock solution (10 g/L), preset to the particular pH value (pH ) 9.00/9.75), was diluted by the carbonate buffer (10 mM) of the corresponding pH value to 0.2 g/L peptide concentration. The volume dilution was approximately 2%. Then the measurements were carried out in a quartz cell with cell length of 0.1 cm. Spectra were smoothed with a convolution length of 15 before the CDSSTR calculations.
Crystal Growth & Design, Vol. 9, No. 5, 2009 2399 Thermogravimetric Analysis for Binding Affinity Estimation. Thermogravimetric analysis (TGA) was performed on a NETZSCH TG 209 F1 instrument in artificial air atmosphere (N2/O2 ) 80/20). The instrument provided a resolution within 0.1-µg mass change. The samples were heated from 20 to 1000 °C at a rate of 10 K/min. Samples for TGA were prepared in the following way: Vaterite was prepared according to ref 39, and calcite and aragonite were prepared according to ref 40. Fifteen milligrams of vaterite, aragonite, and calcite crystals were stirred separately in 0.1 g/L solution of peptide 3 (5 mL, room temperature, 18 h). Subsequently, the crystals were sedimented at a centrifugal force of 8150g (acceleration of gravity g ) 9.81 m s-2) for 70 min, and the supernatant was removed. The crystals were washed with cyclohexane and dried at 65 °C for 3 h. Quantities of 7.2 mg of the calcite, 7.2 mg of the aragonite, and 6.3 mg of the vaterite yield were analyzed separately. The TGA diagrams show that water and cyclohexane were removed completely.
Results and Discussion Qualitative Analysis. The action of the three peptides on CaCO3 crystallization is qualitatively analyzed in gas diffusion experiments, which facilitate precipitation of CaCO3 at increasing pH values starting at approximately pH ) 5-6 and precipitating CaCO3 at approximately pH ) 8.5-9. These experiments facilitate the comparison of the action of the selected peptide additives with literature studies, which mostly utilize the gas diffusion crystallization method, too. Peptides 1 and 3 do not show any distinct effect on CaCO3 crystals in gas diffusion experiments (data not shown), while peptide 2 causes growth inhibition expressed by developed edges, dislocations, and pores as well as surface deposits (Figure 1b-d) in contrast to the crystals grown in the absence of additives (Figure 1a). This indicates that peptide 2 adsorbs on (104) calcite faces. The result is very similar to literature results obtained with two different, highly conserved subdomains of the protein family “Asprich” associated with the prismatic layer of the bivalve Atrina rigida.19 Those subdomains are considered to play important roles in the biomineralization processes in the calcitic layer of the bivalve by the inhibition of the growth of calcite. In this view, it is proximate that peptide 2 inhibits calcite growth, too. The effect of all three peptides on CaCO3 crystallization is to be studied in more detail by means of the quantitative crystallization assay. Quantitative Analysis. The time development of the amount of free calcium ions detected by the calcium ion selective electrode in comparison to the dosed amount of calcium ions in carbonate buffer is illustrated for pH ) 9.00 and 9.75 in Figure 2a,b, respectively. The reference line (black) refers to experiments carried out in the absence of additives, and red, green, and blue lines refer to experiments carried out in the presence of 0.1 g/L of peptides 1, 2, and 3, respectively. The dashed line indicates the amount of calcium ions, which are added. Repetition of the experiments shows that the amount of free calcium ions corresponding to the equilibrium solubility after nucleation as well as the increasing amount of free calcium in solution during the prenucleation stage are reproducible, whereas the time of nucleation and the amount of free calcium at the time of nucleation (maximum before the sharp drop in the amount of calcium) are not. This is understandable as the nucleation rate is determined by a Boltzmann approach, which relates to an activation barrier of nucleation. As discussed in detail in ref 30, the distinct binding of calcium ions in the prenucleation stage can be attributed to the formation of thermodynamically stable CaCO3 clusters, in which the binding strength is high at pH ) 9.00 and finally predominantly calcite is formed, while the binding strength is low at pH ) 9.75 and predominantly vaterite is finally formed (cp. Figure 2a,b).30 After
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Figure 1. SEM images of crystals precipitated in gas diffusion experiments. (a) Typical calcite rhombohedron precipitated in the absence of additives. (b-d) Calcite crystals precipitated in the presence of 1.492 g/L of peptide 2. Deposits can be seen on the calcite surface, which may possibly correspond to amorphous calcium carbonate stabilized by the peptide.
nucleation, the amount of free calcium ions drops to a value corresponding to the particular solubility concentration of the formed phase, which is amorphous CaCO3 (ACC I, presumably calcitic ACC, 3.1 × 10-8 M2) at pH ) 9.00 and ACC II (presumably vateritic ACC, 3.8 × 10-8 M2) at pH ) 9.75.30 Within experimental accuracy, these ACC phases are also formed in the presence of peptide additives, respectively. The peptide additives do not show any influence on the cluster formation equilibria, which would be indicated by an altered slope of the prenucleation time developments of the amount of free calcium ions if compared to the reference experiments. Furthermore, calcium ion binding affinity of the peptide additives, which could be identified by a time-offset of the particular developments illustrated in Figure 2, cannot be observed either. Thus, in the view of the categorization of the action of additives,31 the peptides are no type I/II additives (no adsorption of calcium ions, no influence on cluster formation equilibria). However, peptides 2 and 3 inhibit nucleation, whereas this effect cannot be clearly detected for peptide 1. At pH ) 9.00 (Figure 2a), nucleation occurs in the absence of additives at 6600 ( 250 s, in the presence of peptide 1 at 7100 ( 200 s, in the presence of peptide 2 at 8700 ( 1000 s, and in the presence of peptide 3 at 8700 ( 700 s; at pH ) 9.75, nucleation occurs in the absence of additives at 3900 ( 150 s, in the presence of peptide 1 at 3800 ( 150 s, in the presence of peptide 2 at 4600 ( 100 s, and in the presence of peptide 3 at 4200 ( 50 s (mean value ( maximum deviation). Assuming a stochastic nucleation process, the increasing fluctuation of the time of nucleation with decreasing pH value can be ascribed to the fact that the system stays longer in the supersaturated stage at lower pH values than at higher pH values. This is due to the decreased carbonate ion concentration in the carbonate/bicarbonate buffer at pH 9.00 compared to that at pH 9.75, which also causes a later time of nucleation in those systems.30 However, it is evident that the amount of free calcium, at which nucleation occurs, is higher for peptides 2
and 3 as compared to the reference experiment or peptide 1 and results in a later time of nucleation. This means that peptides 2 and 3 stabilize the system against nucleation of ACC. Peptide 2 stabilizes the system within approximately 30% at pH ) 9.00 and within approximately 20% at pH ) 9.75, and peptide 3 stabilizes the system within approximately 30% at pH ) 9.00 and within approximately 10% at pH ) 9.75 (if judged by the time of nucleation). The stabilization of the supersaturated system against nucleation can only be explained by the colloidal stabilization of clusters31 since free calcium ions are not bound by the peptides within experimental accuracy. At the same time, it is obvious that the stabilization of the system by the peptide additives against nucleation is better at pH ) 9.00 than at pH ) 9.75. Figure 3 illustrates wide-angle X-ray (WAXS) diffractograms obtained from precipitated particles approximately 1.5 h after ACC nucleation. Figure 3a illustrates the WAXS diffractogram obtained from particles precipitated in the absence of additives at pH ) 9.00. The diffraction pattern complies with database calcite patterns, while traces of vaterite (most intense vaterite signals indicated by arrows, cp. also Figure 3b) are detectable. At pH ) 9.75 and in absence of additives (Figure 3b), the diffraction pattern complies with vaterite and traces of calcite (most intense calcite signals indicated by arrows, cf. also Figure 3a). The formation of calcite particles is not due to ripening along Ostwald’s rule of stages of vaterite crystals, because calcite crystals can already be detected shortly after the nucleation event by means of TEM and ED (data not shown). In the presence of all three peptide additives, almost phasepure vaterite is obtained at both pH ) 9.00 and 9.75 as illustrated in Figure 3c,d. A novel signal, which cannot be assigned to calcite, aragonite, or vaterite (indicated by the fine arrow in Figure 3d), is detectable in diffractograms obtained from particles precipitated at pH ) 9.75 in the presence of peptides 1 and 2. The assignment of this signal is not possible, since there are no
Influence of Peptides on CaCO3 Precipitation
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Figure 3. WAXS diffractograms obtained from precipitated particles approximately 1.5 h after nucleation in the absence of additives at pH ) 9.00 (a) and 9.75 (b). Arrows indicate most intensive vaterite signals in (a) and most intensive calcite signals in (b). (c, d) Magnification of the most intensive vaterite signal in WAXS diffractograms obtained from particles precipitated 1.5 h after nucleation in the presence of peptide additives as indicated. Bold arrows indicate the position of the most intensive calcite signal; the fine arrow indicates a not clearly assignable signal. For explanations, see text. Figure 2. Time development of the amount of free calcium ions measured by a calcium ion selective electrode in carbonate buffer at pH ) 9.00 (a) and 9.75 (b). Multiple repetitions are shown. The dotted line gives the amount of calcium ions, which are added. “Reference” (black) refers to experiments in the absence of peptide additives, and colored lines refer to experiments in the presence of 0.1 g/L peptides as indicated. Dashed-dotted lines separate under- and supersaturated areas of the diagrams. The sharp drop in nfree(Ca2+) indicates nucleation.
further additional unassigned signals. Database comparison with all ionic compounds, which can form in the system, shows that the signal is only close to the most intense signal of CaClOH within ∆2θ ) 0.5°. Nevertheless, strikingly, no traces of calcite can be detected (most intense calcite signal position indicated by bold arrows) in the presence of all three peptides at pH ) 9.75 and in the presence of peptides 2 and 3 at pH ) 9.00, whereas the traces of calcite are significantly reduced in the presence of peptide 1 at pH ) 9.00. These XRD results are confirmed by SEM images of crystals obtained at pH 9.00 1.5 h after nucleation (Figure 4). In the presence of peptides 2 and 3, exclusively vaterite crystals are observed assembling in hexagonal discs which form spherical structures. This motif is also identified in the presence of peptide 1 (and without additives, data not shown), but as proposed by the WAXS spectrum, calcite rhombohedra and even needlelike-shaped crystals of aragonite, which cannot, however, be detected in the diffractogram (cp. Figure 3), can be found. Our previous findings30 suggest that calcitic short-range order is predominantly formed in prenucleation clusters and precipitated amorphous particles at pH ) 9.00, whereas vateritic short-range order is predominantly formed in prenucleation clusters and precipitated amorphous particles at pH ) 9.75, while different amorphous short-range orders have been already discussed in the literature.41-44 Basically, the changes in the precursor structures may be attributed to pHdependent changes in cluster formation thermodynamics,30 and the formation of the crystalline particles may be interpreted as a second nucleation event of the particular amorphous phase in
analogy to two-step nucleation mechanisms postulated for protein crystallization.45 This kind of solid-solid phase transformation has also been recently reported by Pichon et al.46 for the formation of vaterite crystals forming under self-assembled monolayers. Obviously, the suppression of calcite formation correlates the inhibition of nucleation for peptides 2 and 3 at different pH values. The more pronounced inhibition of nucleation by these peptides at pH ) 9.00 compared to that at pH ) 9.75 indicates that the cluster structures forming at pH ) 9.00 and 9.75 differ: At pH ) 9.00, predominantly calcitic clusters form and the system can be better stabilized against nucleation than at pH ) 9.75, at which predominantly vateritic clusters form. This conclusion depends on the assumptions that the peptides also show binding affinity to calcite (i) and that the secondary structure (ii) and net charge (iii) of the peptides do not change significantly in the investigated pH interval (see indicated points below). Peptide 1, however, shows an insignificant effect on the time of nucleation but is also capable of inhibition of calcite formation. This can only be explained by the stabilization of precipitated amorphous intermediates against crystallization, which is proximately also true for peptides 2 and 3. (i) TGA shows that 1.2 wt % organics are bound to vaterite, 1.5 wt % organics are bound to calcite, and 2.6 wt % organics are bound to aragonite. This shows that peptide 3 exhibits binding affinity to all polymorphs according to vaterite < calcite < aragonite. (ii) We performed CD measurements on the peptides at the investigated pH values, and it is obvious that the secondary structural features of the peptides change only insignificantly in between the investigated pH values. In Table 1, the results of the structure analysis by means of CD spectroscopy are shown. The structural composition of the peptides was calculated by the CDSSTR method, which provides sufficient accuracy as the fit parameter NRMSD indicates. The calculations show that
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Figure 4. SEM images of the crystals obtained in the presence of the peptides at pH ) 9.00 (peptide 1 left, peptide 2 middle, peptide 3 right). The morphology of the vaterite crystals (spherical polycrystals) corresponds to the morphology found in the absence of the peptides (data not shown). However, formation of calcite is suppressed in the case of peptides 2 and 3, whereas minor amounts of calcite are detectable in the case of peptide 1 (red circles). Also, needlelike crystals are detectable in the presence of peptide 1 (green circles), a morphology often observed for aragonite. However, aragonite cannot be detected by means of WAXS. Cf. also Figure 3. Scale bars: 3 µm (peptides 1 and 2) and 2 µm (peptide 3). Table 1. Results of the Structure Analysis of the Peptides by Means of CD Spectroscopy Using CDSSTR Calculationsa
peptide 1 peptide 2 peptide 3
pH
R-helices
β-strands
turns
unordered
NRMSD
9.00 9.75 9.00 9.75 9.00 9.75
0.03 0.03 0.02 0.03 0.04 0.04
0.27 0.23 0.29 0.25 0.25 0.26
0.15 0.15 0.18 0.15 0.16 0.16
0.54 0.57 0.49 0.55 0.54 0.53
0.035 0.027 0.027 0.030 0.019 0.024
a Different kinds of helices and strands are combined to R-helices and β-strands, respectively. The fit parameter normalized root mean square deviation (NRMSD) is stated.
the proportions of structural elements in peptides 1 and 2 are nearly unaltered at both pH values; merely β-strands and turns (peptide 2) increase slightly at the expense of unordered parts when the pH value is raised to pH ) 9.75. In contrast, the structure of peptide 3 is completely unchanged at both pH values. As expected, the buffers do not show any effect in the absence of additive (data not shown). (iii) We calculated the net charge of the peptides using the freeware SEDNTERP.47 The program gives the isoelectric points (peptide 1, pH 10.40; peptide 2, pH 10.67; peptide 3, pH 10.40) and the net charge pH development. The net charge changes insignificantly from 1.9 (pH ) 9.00) to 1.5 (pH ) 9.70) for peptide 2 and from 1.0 (pH ) 9.00) to 0.7 (pH ) 9.70) for peptides 1 and 3. Altogether, the experiments show that, depending on the peptide sequence, different binding affinities to CaCO3 polymorphs can be obtained. Differently pronounced binding affinities to the different polymorphs then facilitate the preferential formation of the polymorph, which is bound weakest while subtle charge and structural changes do not seem to be of relevance. This shows that polymorph switch can be achieved by additives, which show binding affinity to CaCO3 polymorphs, which are not formed subsequently, and, moreover, that prenucleation stage clusters show different structures, too. This also explains why all three peptides, which show strong binding to aragonite,25 do not nucleate aragonite, but inhibit calcite nucleation in coincidence with a report by Evans et al. on AP7.48 This result also shows that phage display techniques, which are very successful to identify active peptides for the catalysis of mineral formation under conditions at which they would normally not form,49 fail for the direct polymorph control of CaCO3 and likely also other minerals. This finding has deep consequences for understanding biomineralization processes and gives a novel basis for experiments utilizing biological peptide sequences and quantitative analysis of their action on mineralization processes. In the end, comparison of the qualitative and
quantitative crystallization assays shows that the qualitative gas diffusion method may not be able to display weak additive action, which is apparent in the quantitative assay. We attribute the formation of calcite and superposition of the additive action in gas diffusion experiments to strong and unforeseeable pH changes and significant amounts of ammonia present in the system, since the characterization of stable prenucleation CaCO3 clusters shows delicate pH dependency.30 Altogether, the comparison of both assays shows that gas diffusion experiments are a rather bad choice for quantitatively analyzing the crystallization of CaCO3 in the presence of additives. In contrast, the applied quantitative crystallization assay can even detect subtle differences between molecules acting as crystallization additives and can serve as a good test for polymorph control as well as its deeper understanding, a still unresolved question in biomineralization. Acknowledgment. Financial support by the German Science Foundation priority program 1117 “Principles of Biomineralization” and the Max Planck Society is gratefully acknowledged. We thank Katarina Ostwald (MPI) for her support with the preparation of the peptides.
References (1) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989. (2) Mann, S. Biomineralization and biomimetic materials chemistry. J. Mater. Chem. 1995, 5 (7), 935–946. (3) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: Oxford, 2001; p 216. (4) Weiner, S.; Addadi, L. Design strategies in mineralized biological materials. J. Mater. Chem. 1997, 7 (5), 689–702. (5) Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Control of crystal phase switching and orientation by soluble mollusc-shell proteins. Nature 1996, 381 (6577), 56–58. (6) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Control of aragonite or calcite polymorphism by mollusk shell macromolecules. Science 1996, 271 (5245), 67–69.
Influence of Peptides on CaCO3 Precipitation (7) Yu, S. H.; Co¨lfen, H. Bio-inspired crystal morphogenesis by hydrophilic polymers. J. Mater. Chem. 2004, 14 (14), 2124–2147. (8) Xu, A. W.; Ma, Y. R.; Co¨lfen, H. Biomimetic mineralization. J. Mater. Chem. 2007, 17 (5), 415–449. (9) Meldrum, F. C.; Co¨lfen, H. Controlling mineral morphologies and structures in biological and synthetic systems. Chem. ReV. 2008, 108 (11), 4332–4432. (10) Blank, S.; Arnoldi, M.; Khoshnavaz, S.; Treccani, L.; Kuntz, M.; Mann, K.; Grathwohl, G.; Fritz, M. The nacre protein perlucin nucleates growth of calcium carbonate crystals. J. Microsc. (Oxford, UK) 2003, 212, 280–291. (11) Fu, G.; Qiu, S. R.; Orme, C. A.; Morse, D. E.; De Yoreo, J. J. Acceleration of calcite kinetics by abalone nacre proteins. AdV. Mater. 2005, 17 (22), 2678–2683. (12) Mann, K.; Siedler, F.; Treccani, L.; Heinemann, F.; Fritz, M. Perlinhibin, a cysteine-, histidine-, and arginine-rich miniprotein from abalone (Haliotis laeVigata) nacre, inhibits in vitro calcium carbonate crystallization. Biophys. J. 2007, 93 (4), 1246–1254. (13) Michenfelder, M.; Fu, G.; Lawrence, C.; Weaver, J. C.; Wustman, B. A.; Taranto, L.; Evans, J. S.; Morsel, D. E. Characterization of two molluscan crystal-modulating biomineralization proteins and identification of putative mineral binding domains. Biopolymers 2003, 70 (4), 522–533. (14) Samata, T.; Hayashi, N.; Kono, M.; Hasegawa, K.; Horita, C.; Akera, S. A new matrix protein family related to the nacreous layer formation of Pinctada fucata. FEBS Lett. 1999, 462 (1-2), 225–229. (15) Treccani, L.; Mann, K.; Heinemann, F.; Fritz, M. Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals. Biophys. J. 2006, 91 (7), 2601–2608. (16) Gotliv, B. A.; Kessler, N.; Sumerel, J. L.; Morse, D. E.; Tuross, N.; Addadi, L.; Weiner, S. Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida. ChemBioChem 2005, 6 (2), 304–314. (17) Sarashina, I.; Endo, K. Primary structure of a soluble matrix protein of scallop shell: Implications for calcium carbonate biomineralization. Am. Mineral. 1998, 83 (11-12), 1510–1515. (18) Tsukamoto, D.; Sarashina, I.; Endo, K. Structure and expression of an unusually acidic matrix protein of pearl oyster shells. Biochem. Biophys. Res. Commun. 2004, 320 (4), 1175–1180. (19) Collino, S.; Kim, I. W.; Evans, J. S. Identification of an “acidic” C-terminal mineral modification sequence from the mollusk shell protein Asprich. Cryst. Growth Des. 2006, 6 (4), 839–842. (20) Kim, I. W.; Giocondi, J. L.; Orme, C.; Collino, S.; Evans, J. S. Morphological and kinetic transformation of calcite crystal growth by prismatic-associated asprich sequences. Cryst. Growth Des. 2008, 8 (4), 1154–1160. (21) Kim, I. W.; Darragh, M. R.; Orme, C.; Evans, J. S. Molecular “tuning” of crystal growth by nacre-associated polypeptides. Cryst. Growth Des. 2006, 6 (1), 5–10. (22) Walters, D. A.; Smith, B. L.; Belcher, A. M.; Paloczi, G. T.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Modification of calcite crystal growth by abalone shell proteins: An atomic force microscope study. Biophys. J. 1997, 72 (3), 1425–1433. (23) Collino, S.; Evans, J. S. Structural features that distinguish kinetically distinct biomineralization polypeptides. Biomacromolecules 2007, 8 (5), 1686–1694. (24) Evans, J. S.; Samudrala, R.; Walsh, T. R.; Oren, E. E.; Tamerler, C. Molecular design of inorganic-binding polypeptides. MRS Bull. 2008, 33 (5), 514–518. (25) Belcher, A. M. In Biomineralization; Ba¨uerlein, E., Ed.; Wiley-VCH: Weinheim, Germany, 2000; p 247. (26) Collino, S.; Kim, I. W.; Evans, J. S. Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7. Biochemistry 2008, 47 (12), 3745–3755. (27) Kitano, Y.; Hood, D. W.; Park, K. Pure aragonite synthesis. J. Geophys. Res. 1962, 67 (12), 4873–4874. (28) Xu, A. W.; Dong, W. F.; Antonietti, M.; Co¨lfen, H. Polymorph switching of calcium carbonate crystals by polymer-controlled crystallization. AdV. Funct. Mater. 2008, 18 (8), 1307–1313.
Crystal Growth & Design, Vol. 9, No. 5, 2009 2403 (29) Neira-Carrillo, A.; Fernandez, M. S.; Retuert, J.; Arias, J. L. Effect of the crystallization chamber design on the polymorphs of calcium carbonate using the sitting-drop method. Mater. Res. Soc. Symp. Proc. 2004, EXS-1, 321–326. (30) Gebauer, D.; Vo¨lkel, A.; Co¨lfen, H. Stable prenucleation calcium carbonate clusters. Science 2008, 322 (5909), 1819–1822. (31) Gebauer, D.; Co¨lfen, H.; Verch, A.; Antonietti, M. The multiple roles of additives in CaCO3 crystallization: A quantitative case study. AdV. Mater. 2009, 21 (4), 435–439. (32) Carpino, L. A.; Elfaham, A.; Minor, C. A.; Albericio, F. Advantageous applications of azabenzotriazole (triazolopyridine)-based coupling reagents to solid-phase peptide-synthesis. J. Chem. Soc., Chem. Commun. 1994, (2), 201–203. (33) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical Approach; Oxford University Press: New York, 2000. (34) Compton, L. A.; Johnson, W. C. Analysis of protein circular-dichroism spectra for secondary structure using a simple matrix multiplication. Anal. Biochem. 1986, 155 (1), 155–167. (35) Manavalan, P.; Johnson, W. C. Variable selection method improves the prediction of protein secondary structure from circular-dirchroism spectra. Anal. Biochem. 1987, 167 (1), 76–85. (36) Sreerama, N.; Woody, R. W. Estimation of protein secondary structure from circular dichroism spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 2000, 287 (2), 252–260. (37) Whitmore, L.; Wallace, B. A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 2004, 32, W668–W673. (38) Whitmore, L.; Wallace, B. A. Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers 2008, 89 (5), 392–400. (39) Richter, A.; Petzold, D.; Hofman, H.; Ullrich, B. Production, properties and application of calcium carbonate powders 0.1. Production of calcium carbonate by precipitation from solutions. CHEMTECH 1995, 47 (6), 306–313. (40) Nebel, H.; Epple, M. Continuous preparation of calcite, aragonite and vaterite, and of magnesium-substituted amorphous calcium carbonate (Mg-ACC). Z. Anorg. Allg. Chem. 2008, 634 (8), 1439–1443. (41) Addadi, L.; Raz, S.; Weiner, S. Taking advantage of disorder: Amorphous calcium carbonate and its roles in biomineralization. AdV. Mater. 2003, 15 (12), 959–970. (42) Hasse, B.; Ehrenberg, H.; Marxen, J. C.; Becker, W.; Epple, M. Calcium carbonate modifications in the mineralized shell of the freshwater snail Biomphalaria glabrata. Chem.-Eur. J. 2000, 6 (20), 3679–3685. (43) Lam, R. S. K.; Charnock, J. M.; Lennie, A.; Meldrum, F. C. Synthesisdependant structural variations in amorphous calcium carbonate. CrystEngComm 2007, 9 (12), 1226–1236. (44) Politi, Y.; Levi-Kalisman, Y.; Raz, S.; Wilt, F.; Addadi, L.; Weiner, S.; Sagi, I. Structural characterization of the transient amorphous calcium carbonate precursor phase in sea urchin embryos. AdV. Funct. Mater. 2006, 16 (10), 1289–1298. (45) Galkin, O.; Pan, W.; Filobelo, L.; Hirsch, R. E.; Nagel, R. L.; Vekilov, P. G. Two-step mechanism of homogeneous nucleation of sickle cell hemoglobin polymers. Biophys. J. 2007, 93 (3), 902–913. (46) Pichon, B. P.; Bomans, P. H. H.; Frederik, P. M.; Sommerdijk, N. A. J. M. A quasi-time-resolved cryoTEM study of the nucleation of CaCO3 under Langmuir monolayers. J. Am. Chem. Soc. 2008, 130 (12), 4034–4040. (47) Philo, J. SEDNTERP-related files. http://www.jphilo.mailway.com/ download.htm. (48) Kim, I. W.; Collino, S.; Morse, D. E.; Evans, J. S. A crystal modulating protein from molluscan nacre that limits the growth of calcite in vitro. Cryst. Growth Des. 2006, 6 (5), 1078–1082. (49) Ahmad, G.; Dickerson, M. B.; Church, B. C.; Cai, Y.; Jones, S. E.; Naik, R. R.; King, J. S.; Summers, C. J.; Kroger, N.; Sandhage, K. H. Rapid, room-temperature formation of crystalline calcium molybdate phosphor microparticles via peptide-induced precipitation. AdV. Mater. 2006, 18 (13), 1759–1763.
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