Crystal Growth of Calcite Mediated by Ovalbumin and Lysozyme

Article pubs.acs.org/crystal

Crystal Growth of Calcite Mediated by Ovalbumin and Lysozyme: Atomic Force Microscopy Study Kang Zhao,† Meng Wang,† Xiaoqiang Wang,† Congmeng Wu,† Hai Xu,*,† and Jian R. Lu*,‡ †

State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580, P. R. China ‡ Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Schuster Building, Manchester M13 9PL, United Kingdom S Supporting Information *

ABSTRACT: Ovalbumin and lysozyme are two major egg white proteins and putatively related to the formation of the mammillary layer of eggshells. In this work, we have investigated their influences on the morphology and growth kinetics of hillocks at the molecular scale using fluid-cell atomic force microscopy. Our studies identified two roles for ovalbumin, favoring the formation of amorphous calcium carbonate−protein clusters on terrace surface and accelerating the step growth kinetics via reduction of the energy barrier for ion attachment to crystal steps. The two effects are intimately linked to the inherent characteristics of ovalbumin, i.e., being acidic and amphiphilic. In contrast, lysozyme as a basic protein did not induce the formation of any moldable transient phases. Instead, it interacted with step edges and pinned them, leading to step bunching and even step advancement stop at higher concentrations. These roles and their associated interactions on the molecular scale are related to the macroscopic features of eggshells and provide a reliable basis for further investigation into their influences in more complex systems mimicking native biological environment.

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INTRODUCTION

Ovalbumin and lysozyme are major egg white proteins (representing ca. 54% and ca. 3.5%), and their intramineral localization is limited to the mammillary cone layer of the eggshell.8−10 On the other hand, during the initial stage of shell formation when the mammillary cones are typically generated, the two proteins are abundant in the uterine fluid, where the shell calcification is taking place. Thus, the two proteins are most likely implicated in the formation of the mammillary cone layer. In previous studies, we have demonstrated that the two proteins favor the formation of particular phases (e.g., moldable transient phases ACC and vaterite or stable calcite) and modify crystalline calcite habits at the macroscopic level.11−13 However, there is still need for fundamental and direct observations of their effects at the molecular level in the presence of the two proteins. In this study, we have applied AFM to follow their influences on the development of hillocks and steps on the calcite (104) face under in situ solution environment and tried to link the observed microscopic alterations to the macroscopic features of eggshells. The knowledge gained from this work is useful to devise new routes to the controlled synthesis of complex composites.

Biominerals are ubiquitous in nature. They often possess complex morphologies, well-ordered microstructures, and unusual properties and functions and are formed under milder conditions than those currently deployed for producing manmade counterparts. Material engineers and scientists have long been encouraged to learn from nature by mimicking biomineralization processes for the fabrication of advanced materials. One of major challenges toward achieving this goal is to understand the underlying mechanisms associated with different biomineralization processes. Soluble and insoluble biopolymers have been revealed to play pivotal roles in the regulation of biomineral formation, presumably via molecular recognitions and interactions at organic−inorganic interfaces.1,2 Despite considerable macroscopic investigations on biomineralization mechanisms, the direct observation of the interfacial effects at the molecular scale and the fundamental interfacial interactions remain challenging. Atomic force microscopy (AFM) is a valuable tool in addressing these issues because it is capable of in situ observation of the advancement or retreat, kinetics, and morphology of an individual step in a flow-through environment during crystal growth or dissolution, thus revealing stepspecific interactions at the interface.3−7 By using this technique, many small organic molecules, e.g., amino acids and short synthetic peptides, have been shown to influence the step advancement and modify the morphology of growth hillocks. © 2013 American Chemical Society

Received: December 12, 2012 Revised: March 8, 2013 Published: March 12, 2013 1583

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flow rate. The protein-bearing growth solution was then maintained for ca. 10 min to ensure reequilibration, followed by capturing images. Step velocity, ν, was estimated by the angle method proposed by Teng et al.19 Both obtuse (positive) and acute (negative) step velocities were measured on spiral hillocks after the equilibration with each growth solution was well established and the step advancement reached a steady state. Briefly, the scan angle was adjusted to orient the step direction ([4̅41]) parallel to the y-axis. The y-axis scan direction (slow scanning) was then disabled when the tip reached the hillock apex. Step velocities were determined by measuring step displacement over time using the images collected with the y-axis scan direction disabled. These images recorded the movement of steps as the evolution of individual points at step edges over the imaging area as

EXPERIMENTAL SECTION

Crystal Substrate and Solution Preparation. The low index {104} faces of calcite have the largest interplanar spacing and the lowest surface energy.14 As a result, fresh calcite {104} faces were easily obtained by using a razor blade to cleave a single large crystal of optical-quality Iceland spar (Ward’s Scientific, Chihuahua, Mexico), generating rhombohedral calcite fragments with six crystallographically equivalent {104} faces. The cleaved fragments with dimensions of approximately 2 × 2 × 1 mm3 were handled with tweezers to avoid any contamination by skin oil and a jet of N2 was applied to remove small particles and debris from the surfaces. Supersaturated CaCO3 solutions were prepared immediately before use by dissolving reagent-grade calcium chloride (CaCl2·H2O) and sodium bicarbonate (NaHCO3) in Milli-Q water (resistivity of 18 MΩ·cm). The ionic strength of growth solutions was fixed within a narrow range of 0.105−0.111 M using reagent-grade NaCl. The ratio of aCa2+/aCO32− was forced to 1.04 ± 0.01 by adjusting the amount of NaHCO3 and CaCl2·H2O. The supersaturation (σ) is defined by ⎛ aCa 2 +aCO 2 − ⎞ 3 ⎟⎟ σ = ln⎜⎜ K sp ⎝ ⎠

(1)

where “a” refers to the actual ion activity calculated from the numerical code PHREEQC15 and “Ksp” is the equilibrium solubility product of calcite at zero ionic strength. Because the growth mechanism is dependent on the degree of supersaturation, the value of σ in this study was controlled at about 0.8 so that the spiral growth from dislocations is the primary mode. Ovalbumin and lysozyme from chicken egg white were purchased from Sigma-Aldrich and directly used without further purification. The growth solutions containing protein were filtered through a 0.22 μm filter, with protein concentrations varied from nano- to micromoles. The pH values of all the solutions were adjusted to 8.50 ± 0.01 by adding dilute NaOH and HCl aqueous solutions. To minimize shifts in saturation state due to CO2 degassing, solutions were sealed in volumetric flasks immediately upon completing preparation. In Situ AFM Imaging. In situ AFM allows direct observations of calcite growth at the atomic scale. Our AFM experiments were performed on a Nanoscope IVa scanning probe microscope (Digital Instruments, Santa Barbara) outfitted with a commercially available fluid cell. All AFM images were captured at room conditions in contact mode with a J-type scanner (maximum scan area 125 × 125 μm2) and gold-coated Si3N4 tips with a nominal spring constant of 0.06 N/m. The AFM instrument was usually warmed up for 1−2 h before each experiment to reduce the thermal drift of the scanner during data collection. Each AFM characterization began by attaching a freshly cleaved calcite fragment onto a steel puck using cyanoacrylate, which was subsequently mounted on top of the scanner, and then the O-ring in the AFM fluid cell was placed on top of the calcite (104) face and a tight seal was formed between the cell and the crystal surface. After the substrate surface was imaged in air to locate a relatively flat area and to optimize image quality, deionized water was first introduced into the cell by a peristaltic pump to dissolve the topmost layer that might have been damaged during the cleaving process. Following the establishment of the crystallographic orientation of each fragment via the orientations of etch pits developed in deionized water,16,17 the input solution was changed to the control growth solution (without proteins). To ensure that the growth was controlled by surface processes rather than diffusion and that saturation states in the cell were as close to those of the input solutions as possible, a flow rate of 0.6 mL/min was used. To capture the morphological evolution of atomic steps and their interaction with macromolecules, scan rates must be rapid. AFM images were collected using scan rates of 5−20 Hz and a resolution of 512 × 512. In order to minimize the force applied to the surface when operating in contact mode, care was taken to continually adjust the set point voltage to the lowest possible value for which tip−crystal contact was still maintained during scanning.18 Once well-defined spiral hillock regions were detected, the growth solution with protein was then introduced to the flow cell at the same

Figure 1. Fluid cell AFM image of a spiral hillock on a (104) face with the y scan direction disabled across the apex of the hillock. shown in Figure 1. Step velocities (v) were calculated on both sides of the hillock using the equation: v=

SrA N tan θ

(2)

where Sr is the scan rate (lines/second), A the scan size, and N the sampling rate (lines/scan, 512 in this study). θ is the angle between the horizontal axis and the apparent step direction on the image of downward scans, as shown in Figure 1.

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RESULTS AND DISCUSSION Effects of Ovalbumin on Growth Morphology and Kinetics. As expected at σ = 0.806, crystal growth occurred predominately at dislocation defects on a (104) cleavage surface, producing step flow that coiled to form spiral hillocks. Data were collected by first locating a single spiral hillock (Figure 2A). The spiral was usually allowed to grow for at least 1 h in the supersaturated solution to ensure its quality and stability. The spiral hillock is composed of two distinct pairs of step edges, the obtuse steps and the acute ones, both of which are symmetrically related by a c-glide plane. The two obtuse steps ([481̅]+ and [4̅41]+), which form an obtuse angle with respect to the cleavage plane, are crystallographically equivalent, as are the two acute ones ([481̅]− and [44̅ 1]−), which form an acute angle with respect to the cleavage plane.3,20 The differences in the orientation of the exposed carbonate groups lead to nonequivalent step edge geometries and kink site structures between the obtuse and acute steps (Figure 4B). As shown in Figure 2A,B,G, two obtuse steps are on the upper part and two acute ones on the lower side, and all of the AFM images shown in this study were oriented in such a way for convenience except for the AFM image in Figure 1. Subsequently, the input solution was switched to the proteinbearing growth solutions with the fixed supersaturation (σ = 0.806), and after flowing for ca. 10 min, the AFM tip was 1584

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from 1 to 100 nM (Figure 2B−D). As the protein concentration was increased to 1 μM, surface roughening occurred over the obtuse steps whereas the acute steps remained almost unaffected except for a few rounded deposits (ca. 50 nm in diameter). As the exposure time further increased at this concentration, there was no apparent alteration in the hillock morphology. At the higher concentration of 10 μM, surface roughening occurred on both obtuse and acute directions (Figure 2F). Kim et al. observed similar surface roughening on the calcite (104) face during the crystal growth, in the presence of acidic protein fragment (fragment-2).21 They attributed this phenomenon to deposit formation on terrace surfaces. In fact, rounded deposits or aggregates on crystal surfaces have been widely observed in the presence of acidic peptides and proteins and were all presumed to be mineral−organic composites.21−25 In this study, we believe the observed surface roughening arose from the formation of similar dense deposits on terraces. Although the nature or the composition of these deposits remains elusive, their formation is possibly related to the acidic nature of ovalbumin. Specifically, Ca2+ ions accumulate around ovalbumin by complexation with acidic side groups, leading to the formation of ovalbumin−CaCO3 aggregates. Under the present experimental conditions, the calcite (104) surface is positively charged (the point of zero charge is pH 9.5), favoring the subsequent deposition of these aggregates on terrace surfaces.26 On the other hand, it is also possible that ovalbumin molecules first bind onto terrace surfaces due to electrostatic interaction, followed by Ca2+ capturing and the formation of protein−mineral aggregates. Note that these deposits may represent amorphous calcium carbonate (ACC)−ovalbumin composites, in light of their circular morphologies23 and the evident selected area electron diffraction (SAED) analysis as performed in our previous work.13 Since the long-range electrostatic interaction rather than chemical forces dominates during the formation of these surface deposits, their interaction with terraces should be weak and the surface deposits might be readily removed. Thus, we performed continuous scanning and imaging of the same area after the establishment of surface roughening. To minimize the force applied to the surface during this process, great care was taken to continually adjust the set point voltage to the lowest possible value, for which tip−crystal contact was still maintained during scanning. Despite this meticulous action, the surface deposits could be completely swept away by the AFM tip within the 17 min continuous scanning (Figure 3),

Figure 2. Steady-state growth hillocks formed on a freshly cleaved calcite (104) surface in the supersaturated solution (σ = 0.806). (A) Negative control without additive and after the addition of (B) 1 nM, (C) 10 nM, (D) 100 nM, (E) 1 μM, and (F) 10 μM ovalbumin. (G) Schematic cross section of the (104) terrace showing step geometry. To get steady-state spiral hillocks, AFM image capturing was performed after the introduction of the protein-bearing growth solution for ca. 10 min.

engaged onto the calcite surface. The range of ovalbumin concentrations increased from 1 nM to 10 μM. Little change in step morphology was observed at the concentrations ranging

Figure 3. Sequential AFM images showing the elimination of surface roughening by continuous scanning: (A) 0 min, (B) 1 min, (C) 2 min, (D) 3 min, (E) 4 min, (F) 5 min, (G) 6 min, (H) 8 min, (I) 12 min, and (J) 17 min. To ensure the surface roughening establishment, the calcite (104) face was exposed for ca. 10 min to the flowing ovalbumin-bearing growth solution (1 μM) before the tip engagement. 1585

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confirming that the interaction between the deposits and terrace surface was indeed weak. Furthermore, the removal of the deposits on the obtuse−obtuse corner region occurred in the final stage, reminiscent of our previous work in which the deposition was observed to be first initiated at the same region, followed by their propagation along the obtuse steps,13 indicating that relatively higher interfacial energy was associated with this corner area. On the other hand, as a result of weak interaction, these surface deposits did not affect the advancement of the underlying steps, and after sweeping these deposits away, the (104) faces again showed their rhombohedral shape (Figure 3J). This feature also allows us to precisely determine the step velocities at this relatively higher concentration in spite of the initial occurrence of serious surface roughening. Note that, in this case, step pinning that usually arises from the binding of additives onto step edges was not observed. As a result, the step advancement was not impeded. Instead, the increase in step velocity was observed, possibly due to the change of interfacial energy induced by the protein, as will be discussed below. When continuous scanning was done on the growth of hillocks immediately after the introduction of 1 μM ovalbumin, there was no presence of surface deposits or the occurrence of surface roughening on terraces (data not shown), an observation highly consistent with the above results. As indicated above, surface roughening predominately occurred on the terrace surfaces along the obtuse direction at the concentration of 1 μM, suggesting a direction-specific deposition of ACC−ovalbumin clusters. This phenomenon is related to the structure of the growth hillocks. As shown in Figure 4, due to differences in the orientation of the exposed

carbonate groups, the crystal surfaces show different geometries and structures between the obtuse and acute directions. The terrace surface along the obtuse direction is more geometrically open, thus with fewer steric constraints on the residence of relatively larger CaCO3−ovalbumin clusters, as opposed to the geometrically more constrained terrace surface along the acute direction. Another possible explanation for the different surface roughening is that ovalbumin molecules might interact more strongly with the surface along the obtuse direction. Elhadj et al. have investigated the effect of polyaspartates (Aspn) on the growth morphology and kinetics of calcite, and their molecular modeling results indicated that, upon adsorption, the energy required to dehydrate acute steps is greater than that at the obtuse steps for longer Aspn peptides (n > 2), thus favoring their binding to the obtuse steps.27 In spite of great differences in sequence and structure between Aspn peptides and ovalbumin, the acidic nature (due to the presence of more acidic residues, 33 Glu and 14 Asp residues versus 20 Lys and 15 Arg residues) and the relatively larger size of ovalbumin (386 residues, dimensions of 7 × 4.5 × 5 nm3)28 make it behave in the same way as the long Aspn peptides, i.e., preferentially interacting with the crystal surface along the obtuse direction. Note that a few surface deposits along the acute direction were observed at this concentration. This phenomenon is due to the nonspecific deposition of ACC− ovalbumin clusters. At higher concentrations such as 10 μM, it is the nonspecific deposition rather than specific interactions that dictated surface roughening. After ca. 10 min of introduction of the protein, we measured the step velocities at different concentrations. Although surface roughening could be eliminated by the tip moving at the concentration of 1 μM, too fast cluster deposition or morphological alterations obstructed the dynamic measurement process at the higher concentration of 10 μM. Figure 5 shows

Figure 5. Step velocity variations as a function of ovalbumin concentration for obtuse and acute steps. Note that v0 is the measured step speed for the control, and v is the measured step speed in the presence of different ovalbumin concentrations.

the step velocity ratio at ovalbumin concentrations up to 1 μM, ν/ν0, where v0 represents the step speed for the negative control and ν is the step speed in the presence of ovalbumin. The addition of ovalbumin obviously accelerated the advancement of both obtuse and acute steps. Furthermore, such an acceleration of growth kinetics was positive concentrationdependent, with ν/ν0 being near-linear to log[concentration of ovalbumin (M)], during the investigated concentration range. Because step pinning did not occur in the case, we ascribe the acceleration of growth kinetics to the reduction of the energy

Figure 4. The kink environment for obtuse and acute steps: (A) top view of the calcite (104) face and (B) side view of the calcite (104) face, viewed from the [010] direction. 1586

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Figure 6. The growth hillock morphology of calcite before and after lysozyme introduction. Negative control without lysozyme (A) and after the addition of 5 μM (B) and 10 μM lysozyme after 10 min (C), 20 min (D), and 50 min (E and F). To get steady-state spiral hillocks, AFM capturing was performed after the introduction of the protein-bearing growth solutions for ca. 10 min.

0.806. This result appears to be consistent with the trend observed by Elhadj et al. Thus, the investigation at the molecular scale revealed that ovalbumin plays two major roles during calcite growth, i.e., favoring the formation of ACC−ovalbumin clusters on step terraces and modifying the step advancement kinetics. The two effects are intimately linked to their inherent characteristics. First, due to rich acidic residues on the protein surface, calcium ions (very small spheres) are readily captured by the protein via complexation with carboxylate groups, eventually leading to the formation of ACC−ovalbumin clusters on terraces. As a transient mineral precursor, ACC is often unstable and quickly transforms into the crystalline state. However, the presence of ovalbumin can considerably stabilize the moldable transient phase and greatly extend its lifespan, thus favoring its morphology reservation in its later crystalline state.12,13,32,33 In fact, the observed tiny ACC−ovalbumin clusters on terraces are reminiscent of the morphology of mamillary caps of eggshells, which contain tiny aggregates of calcite crystal buds34 and have ovalbumin in the mamillary layer across the calcified shell.8 Furthermore, the protein behaves as an anionic surfactant and can minimize the interaction between ions or ion clusters by aggregation, thereby favoring the incorporation of ions into kink sites at step edges and accelerating the step advancement. The acceleration of growth kinetics by ovalbumin is very important when considering the whole process of eggshell formation, which is very quick, typically within 20 h.35 Effects of Lysozyme on Growth Morphology and Kinetics. Compared with ovalbumin, hen egg white lysozyme is a small and basic (isoelectric point of 11.4) protein containing 129 amino acid residues.36 This study revealed that it displayed very different effects on the growth morphology of calcite. Addition of 1 nM−5 μM lysozyme produced little influence on the spiral hillock morphology, in comparison with the negative control (Figure 6A,B and Figure S1 in Supporting Information). The introduction of 10 μM lysozyme induced several significant effects on the hillock morphology (Figure 6C−F and Figure S2, Supporting Information). The most typical observation is that both obtuse and acute steps became roughened via step edge pinning,

barrier for ion attachment to crystal steps. Under the present experimental conditions (σ = 0.806 and the fixed ratio of calcium to carbonate), the step velocity (v) can be described by the following equation v = βω(aCa 2+ − aCa 2+eq )

(3)

where ω is the molecular volume of calcite (6.13 × 10−23 cm3), β is the kinetic coefficient, and (aCa2+ − aCa2+eq) is the difference between actual and the equilibrium activities of the solute. According to the above equation, the velocity enhancement, i.e. v/v0 > 1, signifies that either (aCa2+ − aCa2+eq) or β must increase. The contribution of the former is weak or little at the fixed supersaturation and the increase of v/v0 should be from β/ β0.29 The magnitude of β is controlled by two factors, i.e., the density of kink sites along the step (nk) and the net probability of attachment to a site, which can be written as exp(−Ek/kT), where Ek is an effective barrier to ion attachment at a kink. In the regime of linear kinetics, i.e. v(σ) being linear, nk is constant.29,30 As a result, the increase in β is controlled by the decrease of Ek. This analysis supports the assumption that the presence of ovalbumin accelerates the incorporation of ions into kink sites at step edges by decreasing the energy barrier. Several studies have in fact reported the step velocity increase of calcite in the presence of acidic peptides and proteins.23,29,31 Fu et al. have described this effect as the surfaction mechanism.31 They suggested that, being amphiphilic and negatively charged, acidic proteins might minimize interactions between ions or ion clusters in solution via an aggregation process, thus increasing the ion attachment to the steps. Alternatively, these proteins might transiently adsorb onto step edges like surfactants, also favoring the uptake of ions into the steps. Elhadj et al. have revealed that the step velocity enhancement scales linearly with net negative charges of peptides. For example, the addition of 0.1 μM (Asp)6, and (Asp3-Ser)6-Asp3 and (Asp3-Gly)6-Asp3 induced 15%, 44% and 64% increase in step velocity at the supersaturation of 0.92.29 In spite of differences in the experimental conditions and the molecular dimensions of additives, the net charge of ovalbumin is −12 and the addition of 0.1 μM ovalbumin induced ∼30% increase in step velocity at a slightly different supersaturation of 1587

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At such a high lysozyme concentration, the attached lysozyme molecules at step edges block ion attachment, and the only way the step advances is via growing around the blocking sites. The drop of v/v0 to almost zero signifies that the average spacing between the adsorbed additives is much smaller than the critical length for the step (Lc).3 As Lc is proportional to 1/σ, increasing supersaturation will favor the step advancement in the presence of additives. Similar to ovalbumin, lysozyme is also elevated in the uterine fluid during the initial stage of shell formation. As a basic protein, however, lysozyme shows different effects on calcite growth on the molecular scale. The protein does not induce the formation of transient phases, possibly due to the electrostatic repulsion between the protein and calcium ions. This observation is consistent with our previous macroscopic study showing that lysozyme favors the formation of calcite.11 Furthermore, lysozyme molecules adsorb to step edges and pin them at relatively higher concentrations, thus impeding step advancement, causing step bunching and finally modifying the morphology. Although the two proteins coexist in the uterine fluid, it remains elusive whether there is interplay between these different influences on the crystal growth. Our preliminary experiment indicated that, upon introduction of the mixture of ovalbumin and lysozyme, the former dictated the influence but the latter seemed to enhance the effect. For example, in the mixture of 100 nM ovalbumin and 100 nM lysozyme, surface roughening resulting from the formation of composite aggregates significantly occurred (Figure S3, Supporting Information), although the individual presence of ovalbumin or lysozyme at this concentration or even at 200 nM produced little effect on the hillock morphology. Given the practical significance and high relevance of the biomimetic synthesis of composite biomaterials, further systematic work is under way to investigate how binary mixtures from the two proteins modulate biomineralization.

whereas the above surface roughening, arising from the deposition of protein/mineral aggregates as in the case of ovalbumin, was not observed. In this case, the acute steps were more prone to pinning than the obtuse steps, as opposed to the case of ovalbumin. Ten minutes after introduction of lysozyme, the growth front along the obtuse steps still remained smooth only with the obtuse−obtuse corner being affected, whereas the acute steps were significantly pinned (Figure 6C). As the exposure time was increased to 20 min and thereafter, the obtuse steps did not remain smooth and the obtuse−obtuse corner became rounded, losing the sharp delineation between the two step directions (Figure 6D−F and Figure S2, Supporting Information). Note that, for some hillocks, the acute steps were pinned to a much greater extent, resulting in the obvious occurrence of step bunching (Figure 6F). After 50 min, there was little variation in the hillock morphology. Because the net charge of lysozyme is positive under the present condition, we postulate that the attachment of lysozyme molecules onto the step edges is predominantly driven by the hydrogen bonds between their amide and amine hydrogens and surface carbonates.6 Unlike ovalbumin, once step pinning or bunching was established in the presence of lysozyme, the effect did not disappear under the interference of the moving cantilever tip, possibly due to this short-range interaction. The different degrees of step pinning along the two step types has to do with the geometry of the hillock and the size of adsorbed molecules. As indicated above, due to differences in the orientation of the exposed carbonate groups (Figure 4B), the obtuse step edge is less sterically constrained and thus more accessible to attachment and detachment of lysozyme, while the acute step edge is less open and less accessible to attachment and detachment. Provided that the binding of small and rigid lysozyme molecules onto the step edge is limited by detachment under the present experimental conditions, adsorption onto the acute step edge will be favored. Due to step pinning, the step advancement will be impeded, leading to the deceleration in step velocity, as indicated in Figure 7. Although this decelerating effect was dependent on lysozyme concentration, there was limited inhibition effect (∼20% reduction) at lower concentrations up to 1 μM. At the concentration of 5 μM, ∼50% reduction in step velocity occurred, and at the higher concentration of 10 μM, step advancement effectively stopped due to serious step pinning, with the broad trend consistent with the mechanistic process.

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CONCLUSIONS As key components of egg white proteins, both ovalbumin and lysozyme are thought to take part in eggshell formation, but their exact roles remain speculative. Our AFM study has demonstrated that the two proteins, with distinct isoelectric points, play different roles in mediating the growth of dislocation hillocks on the calcite (104) surface. Ovalbumin shows two effects: induction of the extensive formation of ACC−protein aggregates on terraces, leading to the occurrence of surface roughening and acceleration of the growth kinetics of both obtuse and acute steps by decreasing the energy barrier of ion attachment to crystal steps. These two effects are putatively related to the formation of mammillary caps and the rapid calcification of eggshells. In contrast, as a basic protein lysozyme showed completely different effects. It did not induce the formation of any moldable transient phases. Instead, it interacted with step edges and pinned them, leading to step bunching and even step advancement stop at higher concentrations.

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Figure 7. Step velocity variations as a function of lysozyme concentration for obtuse and acute steps. Note that v0 is the measured step speed for the control, and v is the measured step speed in the presence of lysozyme.

ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org. 1588

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(28) Stein, P. E.; Leslie, A. G. W.; Finch, J. T.; Carrell, R. W. J. Mol. Biol. 1991, 221, 941−959. (29) Elhadj, S.; De Yoreo, J. J.; Hoyer, J. R.; Dove, P. M. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 19237−19242. (30) Chernov, A. A. Theoretical and Technological Aspects of Crystal Growth. In Materals Science Forum; Fornari, R., Paorichi, C., Eds.; Trans Tech Publications: Enfield, NH, 1998; pp 71−78. (31) Fu, G.; Qiu, S.; Orme, C.; Morse, D.; De Yoreo, J. Adv. Mater. 2005, 17, 2678−2682. (32) Pipich, V.; Balz, M.; Wolf, S. E.; Tremel, W.; Schwahn, D. J. Am. Chem. Soc. 2008, 130, 6879−6892. (33) Wolf, S. E.; Leiterer, J.; Pipich, V.; Barrea, R.; Emmerling, F.; Tremel, W. J. Am. Chem. Soc. 2011, 133, 12642−12649. (34) Lakshminarayanan, R.; Kini, R. M.; Valiyaveettil, S. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5155−5159. (35) Arias, J. L.; Fink, D. J.; Xiao, S. Q.; Heuer, A. H.; Caplan, A. I. Int. Rev. Cytol. 1993, 145, 217−250. (36) Diamond, R. J. Mol. Biol. 1974, 82, 371−391.

AUTHOR INFORMATION

Corresponding Author

*Phone: 86-532-86981569 (H.X.), 44-161-2003926 (J.R.L). Email: [email protected] (H.X.), [email protected] (J.R.L). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants No. 21071151) and UK Engineering and Physical Sciences Research Council (EPSRC). H.X. acknowledges the support by Program for New Century Excellent Talents in University (NCET-110735).

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dx.doi.org/10.1021/cg301820w | Cryst. Growth Des. 2013, 13, 1583−1589