Impact of Chiral Molecules on the Formation of Biominerals: A Calcium

Oct 10, 2012 - Impact of chiral molecules on the growth of biominerals: by differential stereochemical matching to the mirror symmetry-related feature...
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Impact of Chiral Molecules on the Formation of Biominerals: A Calcium Oxalate Monohydrate Example Kang Rae Cho,†,‡,§ E. Alan Salter,∥ James J. De Yoreo,§ Andrzej Wierzbicki,∥ Selim Elhadj,† Yu Huang,*,‡ and S. Roger Qiu*,† †

Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94551, United States Department of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, California 90095, United States § Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States ∥ Department of Chemistry, University of South Alabama, Mobile, Alabama 36688, United States ‡

ABSTRACT: Minerals in organisms often exhibit chiral shapes. The physical mechanism by which chiral information is transferred from molecules to crystal morphology is still not well-defined. In this article, we investigate the influence of chiral molecules on the growth of calcium oxalate monohydrate (COM), a mineral phase commonly found in solanacea plants and human kidney stones, by combining in situ atomic force microscopy (AFM) and molecular mechanics modeling. Three synthetic 6-residue linear aspartic acid-rich peptides of L-Asp6, D-Asp6, and L-Asp3−D-Asp3 are used as surrogate molecules, with the first two being an enantiomeric pair. Our observations show that, while both L-Asp6 and DAsp6 modify the growth of COM by interacting with specific steps on existing faces, the effect from the peptide L-Asp3−D-Asp3 on COM growth is minimal. Furthermore, AFM images reveal that the two enantiomers have different binding preferences to steps that are related by mirror symmetry. As a result, growth morphologies with different chiralities emerge. Molecular modeling reveals the structural relationships between the atomic steps and the enantiomers that are responsible for the enantiomer-specific interactions and provides a prediction of relative binding strengths that are consistent with the experimental observations. Our studies support the general principle that the manifestation of gross chiral features in COM crystals grown in the presence of a foreign chiral agent is caused by differential stereochemical matching between the agent and the mirror symmetry-related features present on the growing surfaces.

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INTRODUCTION Of the many remarkable controls exerted over production of biogenic crystals, formation of chiral morphologies offers an excellent example and has served as both a source of inspiration to materials scientists and a subject of research for mineralogists. Biominerals with chiral morphology are often found in marine organisms as well as terrestrial plants.1−9 For example, in marine alga Emiliania huxleyi, calcite crystals manifest themselves as exquisite chiral plates in coccoliths.2,5,8 Similarly, calcium oxalate monohydrate (COM, CaC2O4·H2O) crystals, another common mineral found in nature, also exhibit chiral morphologies in the leaves of some solanacea plants such as tomato and tobacco.7,9 It is widely believed that macromolecules such as proteins, polysaccharides, and peptides play important roles in the reduction in crystal symmetry and the formation of the chiral morphology in vivo.2,7,9 Recent advances further suggest that the acidic parts of biomacromolecules such as aspartic acid-rich domains are the most responsible for directing biomineralization processes such as nucleation, growth, and aggregation.10−25 However, the physical mechanism by which chiral characteristics is transferred from molecules to crystal morphology is still © 2012 American Chemical Society

not well-defined. In vitro studies using calcite have resulted in diverse views of the control mechanism in chiral morphology formation. For example, Orme et al.26 concluded that formation of chiral morphologies in calcite was induced by the change in step edge free energy due to enantiomer-specific binding of amino acids to the acute step edges. Recently, Maruyama et al.27 proposed that L-Asp induced mild chirality in the obtuse steps during growth of calcite by asymmetrically blocking the kink sites. In the present study, we have investigated the influence of chiral molecules on the growth of COM, commonly found in the aforementioned solanacea plants and human kidney stones, by using in situ atomic force microscopy (AFM) and molecular mechanics modeling. COM is a monoclinic crystal with unit cell parameters of a = 9.976 Å, b = 14.588 Å, c = 6.291 Å, and β = 107.05°.28 The shape and size of these crystals depend on the growth conditions such as ionic strength, pH, temperature, and local environment.29−32 While most of the synthetic COM Received: July 6, 2012 Revised: October 1, 2012 Published: October 10, 2012 5939

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of the copolymer are equally manifest with steps that were previously enantiomer-selective or are simply frustrated. We found that both the L- and D-Asp6 enantiomers altered the growth of COM. Although both inhibited the growth of steps in all directions on the (−101) faces, only one step-type was inhibited on the (010) face. Importantly, the chiral effect was only observed on structures related by mirror symmetry. Our results imply that the manifestation of gross chiral effects in COM crystals grown in the presence of a foreign chiral agent is a consequence of differential stereochemical matching between the agent and the mirror symmetry-related (enantiomorphic) features that are present on the growing surfaces.

crystals exhibit habits with three distinct faces, the (−101), (010), and (120), with aspect ratios similar to those shown in Figure 1A, crystal habits with different aspect ratios are also

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EXPERIMENTAL SECTION

Preparation of Peptides. Peptides of L-Asp6, D-Asp6, and L-Asp3− D-Asp3 with purity over 98% (based on HPLC analysis) were obtained from PEPTIDE 2.0 in powder form. All were stored at −20 °C before use. Preparation of COM Seed Crystals. Seed crystals of COM used for this AFM study were grown in vitro by using a gel method. Details of the method are given in ref 29. Preparation of Solutions. Double distilled water was used for making the growth solutions of calcium oxalate monohydrate (CaC2O4·H2O). Supersaturated CaC2O4·H2O solutions with calcium to oxalate ratio = 1 were prepared by mixing CaCl2·2H2O (reagent grade) with K2C2O4 (reagent grade) solutions at equal volumes. Both the CaCl2·2H2O and K2C2O4 solutions included desired amounts of KCl to ensure a fixed ionic strength of 0.05 M.36 The pH of the mixed solution was adjusted to values between 6.94 and 7.02 by adding dropwise NaOH or HCl into the solution before each experiment. For peptide-containing solutions, the desired amounts of concentrated peptide solution (1 mg/50 mL) were first introduced into CaCl2·2H2O solutions before they were mixed with the K2C2O4 solutions. However, this sequence was not necessary as we found that, with the reverse order of mixing, identical results were obtained. The experimental conditions for COM growth under the influence of Asp6 including solute concentration, supersaturation, and impurity concentrations are summarized in Table 1 in detail. The chemical equation for the reaction of COM crystallization is

Figure 1. (A) Optical microscopy image showing the crystal habit of calcium oxalate monohydrate (COM) seed crystal used in AFM investigation, as viewed from the top of the (−101) face. (B) Schematic of crystal habit of COM showing the commonly expressed faces. The mirror plane bisects the (−101) face and is parallel to the (010) face. (C) In situ AFM image of the triangular growth hillocks on the (−101) face. (D) In situ AFM image of the parallelogram-shaped growth hillocks on the (010) face. Images C and D were obtained by the up-scan. Scale bar is 200 and 100 nm for panels C and D, respectively.

reported under other growth conditions.30,33,34 The habits of the seed crystals used in this study are similar to those drawn in Figure 1B, with the (−101) and (010) faces being the objects of this investigation. We chose COM as a good model system to study formation of chiral morphology in minerals because its crystal structure possesses mirror symmetry (space group: P21/ n, the Deganello notation28), with the mirror plane parallel to the (010) face, and it contains two major facets that are strongly contrasting in structure. The (−101) face is calciumrich and electrostatically positive, and the (010) face is oxalaterich and strongly negative.28,35 The mirror plane makes the flat (−101) surface achiral, while the (010) and (010̅ ) surfaces are nonsuperimposable mirror images of each other. For surrogate molecules, we chose three synthetic 6-residue linear aspartic acid-rich peptides: L-Asp6, D-Asp6, and L-Asp3−D-Asp3. The first two are enantiomers, and the third is a copolymer containing 3mers of L-aspartic acid and D-aspartic acid. Although it too is a chiral molecule because of the asymmetry of the N- and Ctermini, the copolymer enables us to test whether the presence of structural elements from both enantiomers eliminates any chiral effects and, if so, whether this is because the interactions

Ca(aq)2 + + C2O4(aq)2 − + H 2O(aq) = CaC2O4 ·H 2O(s) For the calculation of the ion activity in the solution, the multicomponent speciation program Visual Minteq37 was employed, which uses the Davies equation to approximate activity coefficient corrections. The supersaturation (σ) is defined by σ = 1/2 ) where aCa2+ (aOx2−) and aCa2+e (aOx2− ) are the ln(aCa2+aOx2−/aCa2+e aOx2− e e actual and equilibrium calcium (oxalate) activities in the solution. In the concentration range of calcium and oxalate ions used for the experiments, activities of calcium and oxalate ion can be considered to be nearly equal.38 Thus, σ has a rather simplified form, σ = ln(aCa2+/ aCa2+e ) (see Table 1).

Table 1. Summary of Experimental Conditions for COM Growth under the Influence of Asp6a ratio of dissolved Asp6 to Ca2+ (or C2O42−)

a

concentration (μM) (dissolved Ca2+ or C2O42−)

at 2.13 × 10−4 mg/mL Asp6 (0.3 μM)

at 1.42 × 10−3 mg/mL Asp6 (2 μM)

activity (μM) (Ca2+)

activity (μM) (C2O42−)

supersaturation (σ)

130 160 180 200 220 250

2.31 × 10−3 1.88 × 10−3 1.67 × 10−3 1.5 × 10−3 1.36 × 10−3 1.2 × 10−3

1.54 × 10−2 1.25 × 10−2 1.11 × 10−2 1 × 10−2 9.1 × 10−3 8 × 10−3

56.8 69.3 77.6 85.7 93.8 105.8

56.7 69.3 77.5 85.7 93.7 105.7

0.30 0.50 0.62 0.72 0.81 0.93

Experimentally obtained equilibrium activity of calcium ion at which steps on COM crystals neither advanced nor retreated is 41.9 μM. 5940

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In Situ Atomic Force Microscopy. In situ AFM was performed as described previously.18 Briefly, an atomic force microscope (AFM) (Nanoscope IIIa from Digital Instruments, Santa Barbara, CA), commercially available SiN cantilevers (NP-S, Veeco Probes), fluid cell (MTFML, Veeco Probes), and peristaltic pump (Ismatec) were used to perform in situ AFM experiments at room temperature. A seed crystal of COM was glued with inert polyurethane onto a glass coverslip, which was already glued to a metallic AFM specimen disk. The mounted seed crystal was then placed inside the O-ring of the fluid cell. In situ images of the surface of the COM seed crystal were obtained in contact mode, while solution was flowing through the cell. The solution flow rates were adjusted such that, for a given supersaturation, the step velocity did not change with flow rate, i.e., growth was controlled by surface kinetics rather than bulk mass transport. Solution flow rates of about 2 mL/min were found to be adequate. Because the adsorption rate for small peptides onto the COM surface is much faster than the terrace lifetime,39 the flow rate will not affect the adsorption of the chiral molecules onto the crystal face at the chosen flow rate. No correction was made for distortion of images (hillock shape and step orientation), which occurs due to the finite scan rate and nonzero step velocity. Measurements of Step Velocity. The step velocity was obtained by disabling the slow scanning axis and measuring the difference between the apparent step orientation in images collected during upward and downward scans.40 However, at concentrations close to equilibrium, where steps moved very slowly, step velocity was instead obtained by comparing the positions of steps in images collected sequentially. Computational Methods. Modeling surfaces were constructed from the COM crystallographic unit cell28 (P21/n space group) using Cerius2 crystal builder software (4.8.1 ed; Accelrys: San Diego, CA 2003). Flat and stepped (−101) surfaces of unit-cell dimension 6 × 5 (a = 60.66 Å, b = 72.94 Å, γ = 90°) and two layers deep were built. We have assumed the flat regions of the (−101) surface are terminated by layer A, with oxalates lying flat on the surface41 (see Figure 1d of ref 41). Because the actual atomistic nature of the steps on the (−101) surface are unknown, we considered two cases each of the acute and obtuse steps, both with and without oxalates as riser terminations as shown in Figure 2. The L-Asp6 peptide was built with all carboxylates

COM cluster. The central unit cell of the cluster reproduced the experimental COM unit cell atom coordinates with rms deviation = 0.17 Å. Energy minimizations of the COM/L-Asp6 systems were carried out using NAMD 2.7.46 Starting from the beta-sheet conformation, the peptide was manually docked in several different initial orientations (20 or more) on the flat COM surface and in four orientations along the COM step risers. The four step orientations were generated by 180° rotations about and perpendicular to the peptide backbone axis. 3D periodic boundary conditions were imposed to match the 2D surface dimensions of the COM crystal lattice with a vacuum region above the surface (∼80 Å). An implicit water model (ε = 80) was used with a 10 Å cutoff for nonbonded interactions (smoothing on at 9 Å), and the atoms of the crystal lattice were held fixed. Optimizations were followed by short NVT dynamics (10 000 steps, 1 fs/step) successively at 100, 200, and 298 K, and then a second optimization. The dynamical heating phases were carried out to explore more conformations of the peptide at the step and to permit escape from local minima. The energetically most favorable binding orientations, which were determined by the lowest system energy, were selected for further manual manipulation of the peptide geometry, including shifts of the peptide to similar but nonequivalent locations, followed by new energy optimizations. The self-energy of the fixed crystal is not included in the energy evaluation in our model; thus, a comparison of the final energies of the most stable structures directly indicates the relative binding preferences of L-Asp6 and D-Asp6. We are chiefly interested in the binding preference of the L-Asp6 enantiomer for a particular step over the mirror-image step. For such a case, the relative free energy of binding (ΔΔGb) is given by the difference of system energies (E) determined at 0 K; that is, ΔΔGb = ΔΔHb − TΔΔSb ≈ ΔΔEb = E(LAsp6/step) − E(L-Asp6/mirror-image step). We assume a near cancellation of entropies of binding for the two enantiomers (ΔΔSb ≈ 0) as well as a near cancellation of thermal corrections to the enthalpies of binding so that ΔΔHb ≈ ΔΔEb. Of course, in the expansion of the expression for ΔΔEb, the energies of the isolated step and mirror-image step cancel exactly. We note that free energy calculations (which require much more computational resources) may enable one to estimate the relative coverage of the adsorbates at the interested surface step and thus make the connection to the kinetics control of COM growth. However, our approach is adequate to elucidate the differential growth modification caused by the L- and D-Asp6 enantiomers. Such an approach has been demonstrated to be valid in explaining the difference in kinetics control of calcite growth by polyaspartate chains (Aspn, n = 1−6)47 and the single-amino acid enantiomers (L- and D-Asp),26 respectively. Analogously, we are justified in comparing the energies of the enantiomers at the same step: ΔΔGb ≈ ΔΔEb = E(L-Asp6/step) − E(D-Asp6/step). As the D-Asp6 enantiomer was not explicitly modeled, the energy of D-Asp6 on a particular step was determined by modeling L-Asp6 on the corresponding mirror-image step. Our contention is that the peptide is very flexible and can fairly closely approximate the same coordinations with a step or the mirror-image step, but not without introducing the subtle internal strains that are responsible for stereoselective step inhibition. For this analysis, the simple computational model we have chosen is appropriate and preferable to a more complex one involving dynamics in the presence of explicit solvent molecules and perhaps counterions. Such a complex model would greatly obscure the differential internal strains we expect to find and greatly complicate our ability to extract the key information. Moreover, whether the force-field has the ability to account for such subtle strains or not, dynamical modeling will not remedy it.

Figure 2. Riser terminations of the acute [120] and obtuse [−1−20] steps on the (−101) COM surface. Riser types b and a are with and without the circled oxalates at the riser, respectively. The enantiomorphic acute [1−20] and obtuse [−120] steps are analogously defined. deprotonated and the terminal amine protonated, resulting in a net charge of −6 for the peptide and the system as a whole. The CHARMM22 force-field42 with CMAP dihedral corrections43 was applied to the peptide. For the COM ionic lattice, calcium ions were assigned atomic charges of qCa = +2.0, with nonbonded parameters of ε = −0.1186 kcal/mol and Rmin/2 = 1.6508 Å as determined previously for a bulk ionic crystal containing calcium ions;44,45 oxalate carbon and oxygen parameters were taken from the standard CHARMM22 parameters of the carboxylate moiety of aspartate (atom types CC and OC), with atom charges set to qC = +0.52 and qO = −0.76; waters were assigned standard TIP3P parameters. Although the COM lattice is held fixed in our simulations, this set of parameters for COM was tested by carrying out an energy minimization of an isolated 5 × 5 × 5

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RESULTS The COM seed crystals used in current work had similar habit to that reported in earlier studies,18 but most were twinned along the (−101) face. The optical microscope image of the seed crystal viewed perpendicular to the (−101) face is shown in Figure 1A. For clarity, a schematic is included in Figure 1B to 5941

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display the typical shape of the COM crystal with distinct faces labeled.18 At low to moderate supersaturations, COM grew on steps generated at the dislocation outcrops, like many other solution-grown crystals over the similar supersaturation ranges.18,21,22 No two-dimensional (2-D) nucleation was observed. In general, there were more growth hillocks on the (−101) face than on the (010) face, and at steady state, growth usually originated on a few dominant hillocks with some minor coexisting sources. Representative examples of the growth hillocks on both the (−101) and (010) faces are shown in Figures 1C,D. The (−101) Face. The growth hillocks (Figure 1C) on the (−101) face had a triangular shape that differs from the hexagonal crystal shape (Figure 1A,B). The steps that grew toward the (010), (0−10), (−120), and (−1−20) faces were rarely expressed. However, steps moving toward the [−10−1] direction, which truncate the angles formed by the enantiomorphic [120] and [1−20] steps, were observed. These three expressed steps thus formed the triangular shape of growth hillocks on the (−101) face. The absence of the other four steps may be attributed to their considerably higher growth velocities. For growth in pure solutions, at σ = 0.93, the [−10−1] step moved at about 26 nm/s and was more than 10 times faster than the other two. The apparent orientation of the [−10−1] step line shown in Figure 1C, which is not quite normal to the [−10−1] direction, is just an imaging artifact associated with finite scan rate discussed in the experimental section. Measurements of Step Velocity. The addition of L-Asp6 or D-Asp6 enantiomers drastically altered the growth morphology and kinetics of steps along all directions. The resulting morphology with L-Asp6 exhibited mirror symmetry to that with D-Asp6 (Figure 3A,B). As shown in Figure 3A, under the influence of 0.3 μM D-Asp6, the growth hillock morphology changed from the triangular shape in pure solution shown in Figure 1C to that of an ellipse. In the case of D-Asp6, the major axis of the ellipse (see red inset) was rotated approximately 20° counterclockwise from the [−10−1] direction. In contrast, as displayed in Figure 3B, under the influence of 0.3 μM L-Asp6, the ellipse was tilted in the opposite direction with its major axis rotated clockwise about 20° from the [−10−1] direction (see blue inset). The appearance of the enantiomorphic [−1−20] and [−120] steps, which propagate in the directions opposite to those of the [120] and [1−20] steps in Figure 1C and are thus expressed as part of the ellipses shown in Figures 3A,B, suggests that the step velocities were reduced by the presence of the enantiomers. The resulting serrated step edges along all directions also suggest that the enantiomers pinned the steps. The observed chiral effect on modifying the growth morphology can be attributed to the fact that L-Asp6 and DAsp6 selectively interacted with distinct steps on the existing face. While the D-Asp6 strongly inhibited the [120] and [−120] steps, the L-Asp6 preferentially interacted with the [1−20] and [−1−20] steps. To demonstrate the chiral-selective interaction between the enantiomers and the specific steps, step velocity ratios V[1−20]/ V[120] and V[−1−20]/V[−120] were quantified for growth under the influence of either 0.3 μM D-Asp6 or 0.3 μM L-Asp6 at different solution supersaturations. For all solution conditions, under the D-Asp6 enantiomer, the ratios V[1−20]/V[120] and V[−1−20]/V[−120] (Figure 3C,D, filled triangle) were greater than unity. This indicates that the step velocities along the [120] and the

Figure 3. (A) Representative in situ AFM image showing the effect of 0.3 μM D-Asp6 on the growth of hillocks on the (−101) face after flowing 0.3 μM D-Asp6-containing solution of σ = 0.81 for 23 min. The morphology of these hillocks in pure supersaturated solution is shown in Figure 1C. (B) Image showing the effect of 0.3 μM L-Asp6 on the growth of the same hillocks after following 0.3 μM L-Asp6-containing solution of σ = 0.81 for 21 min. Images (A,B) were collected by the up-scan. Scale bars (A,B) are 200 nm. (C) Plot of V[1−20]/V[120] under the influence of either 0.3 μM L-Asp6 (filled circle) or 0.3 μM D-Asp6 (filled triangle) at different solution supersaturation. (D) Plot of V[−1−20]/V[−120] of either 0.3 μM L-Asp6 (filled circle) or 0.3 μM DAsp6 (filled triangle) at different solution supersaturations.

[−120] directions were smaller than their counterparts, confirming the stronger inhibition of the growth of these two lower steps by the D-Asp6 enantiomer. However, because V[−1−20]/V[−120] is more than twice V[1−20]/V[120], the resulting ellipse was tilted upward as shown in Figure 3A. For the L-Asp6 enantiomer, the ratio of V[1−20]/V[120] and V[−1−20]/V[−120] (Figure 3C,D, filled circle) were both less than unity. This demonstrates that the step velocities of the [1−20] and the [−1−20] steps were smaller than their counterparts and confirms the inhibition of these two upper steps. Moreover, in analogy with D-Asp6, the larger effect on V[−1−20]/V[−120] produced the resulting downward tilt of the ellipse (Figure 3B). These results demonstrate that the selective binding of the two different enantiomers to specific steps induced complementary chiral growth hillocks. Moreover, the larger effect on V[−1−20]/V[−120] than on V[1−20]/V[120] due to the addition of either the L- or D-enantiomer shows both that the enantiomeric selectivity was greatest for the mirror symmetry-related [−1− 20] and [−120] steps and that the magnitude of the chiral modification to the hillock shape was dominated by the peptide-step interaction at those steps. Both enantiomers also inhibited the growth of the [−10−1] step through step pinning, and this contributed in large part to the resulting growth hillock morphology. However, if the waters of hydration are ignored for simplicity, the [−10−1] step preserves the mirror symmetry of the COM crystal lattice, and as expected, there is no differential impact by the two enantiomers on the growth of the [−10−1] step. In truth, there are actually two distinct, enantiomophic [−10−1] steps that differ only by water positions at the step edge. For the two steps, the water positions are related by the (010) mirror plane. One enantiomer will have a stronger affinity for one of the two 5942

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For simplicity, the steps in the mirror-related Type II growth hillocks are labeled the same way as those in the Type I. In general, the step velocity of the ⟨100⟩ steps was faster than that of the ⟨001⟩ steps. The two velocities differed by a factor of ∼1.5 as can be seen by the larger spacing between the ⟨100⟩ steps as compared to the ⟨001⟩ steps (Figure 1D and Figures 4C,H). Furthermore, the growth hillocks on the (010) face originated from rather complicated sources, consisting of multiple screw dislocations, mostly of the Frank−Reed type.41,49 As expected, due to the lack of a mirror plane perpendicular to the (010) face, no chiral effect was observed on the growth hillocks after L- or D-Asp6 was added to the growth solution. However, because the geometric relationship between the peptides and the steps are still enantiomer-specific, the magnitude of the effects for the two peptides differed, with LAsp6 being a slightly more potent modifier for the Type I growth hillocks. Both modestly inhibited growth of the ⟨100⟩ step, causing the step edges to become slightly serrated, and had a very minor effect on the growth of the ⟨001⟩ step. Examples of modified Type I growth hillocks in the presence of 2 μM L-Asp6 and D-Asp6 are shown in Figure 4D,E, respectively. As Figure 4F shows, for equal concentrations of the two enantiomers, L-Asp6 reduced the [−100] step velocity by ∼30%, while D-Asp6 suppressed it by ∼20%, and the normalized step velocities with L-Asp6 were lower than those with D-Asp6 for all supersaturations. These step speed effects are also reflected in their morphologies, which are guided by blue and red curved lines in Figure 4D,E. The effects of 2 μM L-Asp6 or D-Asp6 on the growth of the Type II growth hillocks, which are shown in Figure 4G,I,J, were essentially identical to those observed for the Type I hillocks, except that the relative effects of L- and D-Asp6 were reversed due to the mirror symmetry between the two types of hillocks. For example, on Type II hillocks, the reduction in [−100] step speed caused by D-Asp6 relative to that caused by L-Asp6 exhibited the same ratio as obtained on Type I hillocks for LAsp6 relative to D-Asp6. Thus, although no chiral effects were observed on either Type I or II hillocks individually, when compared to one another, the handedness of the peptides was manifest. Again, the mechanism by which these two enantiomers control the kinetics and morphology of the [−100] step is beyond the scope of this article and is discussed in detail elsewhere.48 Effects of a Racemic Mixture and Copolymer of L- and D-Peptides. To confirm the observation of chiral growth modification by individual enantiomers, additional experiments were carried out in which a racemic mixture containing equal amounts of L- and D-peptides was added to the growth solution. Under these reaction conditions, one would expect that steps may experience interaction with both enantiomers. In fact, this is exactly what was observed on both the (−101) and (010) faces. The resulting morphology of the growth hillocks on the (−101) face under the coaddition of 0.3 μM L-Asp6 and 0.3 μM D-Asp6 solution is shown in Figure 5A,B, with Figure 5A having been collected in pure solution. Steps along all directions were affected almost equally; the [120] and [1−20] steps exhibited nearly identical morphological and kinetic changes. Apparently these two enantiomers acted in concert to inhibit the growth on the (−101) face by independently interacting with the steps they modify individually. Furthermore, because of the inhibition along all step directions, those that previously were unexpressed in pure solution, such as the [010], [0−10],

steps because of the water positions, but by symmetry, E(enantiomer1/step1) = E(enantiomer2/step2) > E(enantiomer1/step2) = E(enantiomer2/step1). Thus, no overall differential effect along the [−10−1] step direction is to be seen. The mechanism by which these two enantiomers control the kinetics and morphology of the [−101] step is beyond the scope of this article and is discussed in detail elsewhere.48 The (010) Face. COM often forms crystals that are twinned along the (−101) plane, as shown by the example in Figure 4A.

Figure 4. (A) Optical microscopy image showing twin COM crystals along the (−101) face with two (010) faces expressed. (B) Schematic of the apparent shape of the (010) face of the upper crystal of the twin crystals and growth hillocks observed in it. One is designated as Type I and the other as Type II growth hillocks, respectively. Representative images of Type I growth hillocks in solution of σ = 0.81 are shown in (C−E) with (C) no peptide, (D) 2 μM L-Asp6 after flowing for 42 min, and (E) 2 μM D-Asp6 after flowing for 42 min. Plots showing the normalized step velocity of the [−100] step of Type I (F) and Type II (G) hillocks on the (010) face under the influence of 2 μM L-Asp6, 2 μM D-Asp6, or 2 μM L-Asp3−D-Asp3 at different solution supersaturations. Representative images of Type II growth hillocks in solution of σ = 0.72 are shown in (H−J) with (H) no peptide, (I) 2 μM L-Asp6 after flowing for 59 min, and (J) 2 μM D-Asp6 after flowing for 29 min. Images C−E and H−J were obtained by the up-scan. Scale bars (C−E and H−J) are 100 nm.

This is the same crystal as that shown in Figure 1A when viewed either perpendicular to the (010) face or to the (0−10) face of its juxtaposed twin. Because of this tendency toward twinning on the (−101) plane, growth hillocks that are mirror reflections of one another, designated here as Type I and Type II (Figure 4B), can both be observed in either of the twinned crystals. These growth hillocks both have the shape of a parallelogram, which for the Type I hillock is bound by the two sets of ⟨100⟩ and the ⟨001⟩ steps (Figure 1D and Figure 4C). 5943

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Table 2, which provides system energies that permit prediction of relative binding energies. For comparison, the energy of Asp6 on the (−101) flat plane is also included. Since the self-energy of the crystal is not included in the energy evaluation, the final energies of the most stable structures directly indicate the relative binding preferences of L-Asp6 and D-Asp6 for the different steps, with a lower energy indicating more favorable binding. In general, the results in Table 2 indicate that binding to simple acute and obtuse steps (type a risers) is more favorable than binding to the flat (−101) surface by >9 kcal/ mol. Also, the results indicate that Asp6 binding to the type b risers is, as expected, less favorable than binding to type a risers because of the presence of the riser oxalate ions. In the case of the obtuse [−120] b riser, L-Asp6 binding is actually less favorable than binding to the flat surface, and during dynamical heating phases in which L-Asp6 is initially placed at the riser base, the peptide travels away from the step.

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DISCUSSION The experimental and modeling results present a consistent picture of L- and D-Asp6 interactions with COM. The results in Table 2 show that L-Asp6 binds preferentially to either type of obtuse [−1−20] step over its mirror-image obtuse [−120] step. The preferences are 1.9 kcal/mol for riser case a and 1.8 kcal/ mol for riser case b. In the case of the b riser, binding is favored at the obtuse [−1−20] step over binding to the flat (−101) surface by about 1.1 kcal/mol, while binding on the flat plane is actually preferred over binding near the base of the obtuse [−120] step. Thus, the results for both a and b type risers are consistent with the strong differential impact observed for growth morphology and kinetics of the obtuse steps, as observed experimentally and shown in Figure 3D. According to the modeling, L-Asp6 binding to the acute [120] step is preferred over binding to the enantiomorphic acute [1− 20] step by about 1.7 kcal/mol for riser type a, Table 2. As previously stated, it is not known which riser type (a or b) is actually expressed. If the acute step with riser type a were expressed, our computational results would imply that a strong differential impact on growth morphology/kinetics should be apparent for the acute steps, which is evidently not the case (Figure 3C). In contrast, our modeling shows only a weak discrimination between the acute type b steps of about 0.2 kcal/ mol, again with L-Asp6 favoring the [120] step. Because the experimental results shown in Figure 3C demonstrate a very weak stereoselective inhibition of the [1−20] step over the [120] step by L-Asp6, we conclude that we are at the limit of our model’s ability to discern such a weak binding preference, but we reach a tentative conclusion that the type b acute step is likely to be the actual one. We add that the oxalates at the acute b riser serve to partially satisfy the coordination of the calcium ions along the step edge, yielding perhaps a more stable structure than the simple acute step (type a). An examination of the binding orientations of L-Asp6 at the type a and b obtuse [−1−20] steps (Figure 6) shows that, evidently, the stereoselective preference of L-Asp6 for both types of obtuse [−1−20] steps over the [−120] steps arises from the ability of the peptide to engage the steps with its carboxylate groups lying along the planes of the upright oxalates and with the alpha-hydrogens (circled) directed up relative to the surface. Indeed, for riser case a, the approaching carboxylates of residues 1 and 3 (gold ovals) mimic the positions of upright oxalates of the upper lattice and stack above other upright oxalates of the lower layers. The

Figure 5. Representative in situ AFM images showing the effects of the racemic mixture of equal amounts of L-Asp6 and D-Asp6 on the growth of hillocks on the (−101) and the (010) faces. (A,B) Morphologies of the hillocks on the (−101) face from solution at σ = 0.81 with (A) no peptide and (B) coaddition of 0.3 μM L-Asp6 and 0.3 μM D-Asp6 after flowing for 50 min. (C,D) Morphologies of the hillocks (Type II) on the (010) face from solution at σ = 0.93 with (C) no peptide and (D) coaddition of 1 μM L-Asp6 and 1 μM D-Asp6 after flowing 22 min. Images A−D were collected by the up-scan. Note that images A and B are rotated by 180° when compared with the originally obtained images. Scale bars (A−D) are 100 nm.

[−120], and [−1−20] steps, were expressed under the influence of the peptides. As a result, steps in the first and second turns of the hillock exhibited a shape that resembled the bulk crystal morphology (see dotted insert in Figure 5B) shown in Figures 1A,B. In contrast to the impact observed on the (−101) face, the effect of coaddition on the (010) hillocks, which is shown in Figures 5C,D, was barely distinguishable from that seen with individual additions (Figures 4H-J) due to the minor level of inhibition on the steps produced by these peptides individually. When the copolymer L-Asp3−D-Asp3 was added to the growth solution, the morphology of the growth hillocks on both the (−101) and the (010) faces showed little or no change. As shown in Figure 4F,G (black squares), for both types of the growth hillocks on the (010) face, the normalized step velocity under the influence of the copolymer remained near unity. This was also true for the normalized step velocity on the (−101) face under the influence of the copolymer. The combination of the L- and D-portions of the copolymer apparently interfered with the peptide−step interaction. Molecular Models of L- and D-Asp6 Binding to Steps and Terraces. In order to understand the stereochemical relationships that lead to the observed enantiomer-specific effects, we performed molecular mechanics modeling of Asp6 binding to the terraces and to the mirror-symmetry related steps on the COM (−101) face. CHARMM22 force field modeling results for the Asp6 enantiomers bound to (−101) stepped surfaces are summarized in Figure 6, which shows the binding orientations of L-Asp6 at obtuse [−1−20] steps, and in 5944

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Figure 6. L-Asp6 bound to the base of the obtuse [−1−20] step with riser types a (left) and b (right). Top and side views. Riser regions are indicated in blue. Some important interactions of the carboxylates and carbonyl oxygens with surface and riser calcium ions are indicated (green lines). Stereoselective inhibition of the steps arises from the compatible approach of the indicated carboxylates (gold ovals and arrows) along the planes of upright oxalates of the lattice with the alpha-hydrogens pointed up (circled). With this approach, favorable interactions with the calcium ions at the riser tops are achieved without introducing significant internal strain in the peptide.

leads to a natural up orientation of the carboxylate carbons (Cγ) suited for COO−···Ca2+ interaction at the top of either riser, without significant introduction of internal strain. Indeed, the dihedral angles d(H−Cα−Cβ−Cγ) of residues 3 and 5 for the near ideal pose shown for L-Asp6 bound to riser type b (Figure 6, right side) are both 61.8°, essentially the optimal value of 61° obtained for the peptide when all nonbonded interactions are turned off. It also follows that the alternate residues, which are away from the step (the even-numbered residues), are in natural down positions for favorable interactions with surface ions without high internal strain. The situation on the (010) face is, of course, quite distinct because there is no mirror symmetry relating any pair of steps on a given hillock. Nonetheless, because twinning across the (−101) plane creates subsegments of the crystal expressing (010) and (0−10) faces, for which the lattices are related by a mirror symmetry, the differences in L-Asp6 and D-Asp6 binding affinity to either the terrace or the steps should be equal and opposite for Type I and Type II hillocks. This in turn implies that the changes in hillock morphology and step kinetics should exhibit the same relationship, as is observed experimentally (Figure 4). Why the effects of both enantiomers are so weak is easily understood from the structure of the (010) face and its steps. As discussed in an earlier study41 (see Figure 7 in ref 41), at both the terraces and the step risers of the (010) face, the oxalate ions extend beyond the planes in which they reside. As a result, the negatively charged oxygen atoms of these oxalates electrostatically repel the carboxylate moieties of the peptides,

Table 2. Energies (kcal/mol) of the Asp6 Enantiomers Bound to Steps on the (−101) Face of COM Crystal L-Asp6

(−101) plane acute [1−20] riser a acute [120] riser a acute [1−20] riser b acute [120] riser b obtuse [−1−20] riser a obtuse [−120] riser a obtuse [−1−20] riser b obtuse [−120] riser b

−46.18 −58.07 −59.75 −50.00 −50.21 −57.74 −55.86 −47.28 −45.47

a

D-Asp6

−46.18a −59.75b −58.07b −50.21b −50.00b −55.86c −57.74c −45.47c −47.28c

a

The (−101) surface has a horizontal mirror plane; hence, the L-Asp6 and D-Asp6 binding energies on the plane are equivalent by symmetry. b Equivalent by symmetry to L-Asp6 on the complementary mirrorimage step. The [120] and [1−20] steps of the same riser type are enantiomorphic. cEquivalent by symmetry to L -Asp6 on the complementary mirror-image step. The [−1−20] and [−120] steps of the same riser type are enantiomorphic.

carboxylate of residue 5 (dashed gold oval) approximates this as well but is impacted by the binding of the C-terminus. For riser b, the carboxylates of residues 3 and 5 cannot take positions of upright oxalates because, of course, those sites are occupied by riser oxalates, but the same plane of approach (arrows) enables interactions with calcium ions at the top and base of the step riser. The up orientation of the alpha-hydrogens of the residues along the step (the odd-numbered residues) is key because it 5945

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from Graduate Study Abroad Scholarship by the Korean Science and Engineering Foundation (KOSEF). We thank the Alabama Supercomputing Authority for computational resources. E.A.S. thanks Keith Battle at the University of South Alabama for assistance in carrying out modeling simulations.

thereby minimizing their binding and thus their impact on growth. The absence of chiral effects on growth hillocks of the (−101) face following the addition of a racemic mixture of LAsp6 and D-Asp6 confirms that the observed chiral modification can only be realized by the stereochemical recognition between steps related by mirror symmetry and structurally matched enantiomers. The minor impact of the L-Asp3−D-Asp3 copolymer on COM growth confirms that, in spite of the free rotations of a flexible peptide, the mere presence of the same number and types of functionalities does not necessarily lead to significant step binding. The chiral center conformations are indeed critical, and it may be the case that the absence of the full sequence of compatible chiral centers for a binding to a particular step is compounded by additional strain arising from the presence of the other chiral centers.

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(1) Hazen, R. M. Sci. Am. 2010, 302, 58. (2) Borman, A. H.; Dejong, E. W.; Huizinga, M.; Kok, D. J.; Westbroek, P.; Bosch, L. Eur. J. Biochem. 1982, 129, 179. (3) Cook, T. A. The Curves of Life; Dover Publications: Mineola, NY, 1979. (4) Mann, S.; Sparks, N. H. C.; Blakemore, R. P. Proc. R. Soc. B 1987, 231, 477. (5) Young, J. R.; Didymus, J. M.; Bown, P. R.; Prins, B.; Mann, S. Nature 1992, 356, 516. (6) Aizenberg, J.; Hanson, J.; Koetzle, T. F.; Leiserowitz, L.; Weiner, S.; Addadi, L. Chem.Eur. J. 1995, 1, 414. (7) Bouropoulos, N.; Weiner, S.; Addadi, L. Chem.Eur. J. 2001, 7, 1881. (8) Didymus, J. M.; Young, J. R.; Mann, S. Proc. R. Soc. B 1994, 258, 237. (9) Levy-Lior, A.; Weiner, S.; Addadi, L. Helv. Chim. Acta 2003, 86, 4007. (10) Mann, S. Nature 1988, 332, 119. (11) Addadi, L.; Moradian, J.; Shay, E.; Maroudas, N. G.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 2732. (12) Berman, A.; Addadi, L.; Weiner, S. Nature 1988, 331, 546. (13) Fu, G.; Qiu, S. R.; Orme, C. A.; Morse, D. E.; De Yoreo, J. J. Adv. Mater. 2005, 17. (14) Fu, G.; Valiyaveettil, S.; Wopenka, B.; Morse, D. E. Biomacromolecules 2005, 6, 1289. (15) Guo, S. W.; Ward, M. D.; Wesson, J. A. Langmuir 2002, 18, 4284. (16) He, G.; Dahl, T.; Veis, A.; George, A. Nat. Mater. 2003, 2, 552. (17) Konya, E.; Umekawa, T.; Iguchi, M.; Kurita, T. Eur. Urol. 2003, 43, 564. (18) Qiu, S. R.; Wierzbicki, A.; Orme, C. A.; Cody, A. M.; Hoyer, J. R.; Nancollas, G. H.; Zepeda, S.; De Yoreo, J. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1811. (19) Shiraga, H.; Min, W.; Vandusen, W. J.; Clayman, M. D.; Miner, D.; Terrell, C. H.; Sherbotie, J. R.; Foreman, J. W.; Przysiecki, C.; Neilson, E. G.; Hoyer, J. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 426. (20) Yamamoto, Y.; Nishimura, T.; Sugawara, A.; Inoue, H.; Nagasawa, H.; Kato, T. Cryst. Growth Des. 2008, 8, 4062. (21) Wang, L. J.; Qiu, S. R.; Zachowicz, W.; Guan, X. Y.; DeYoreo, J. J.; Nancollas, G. H.; Hoyer, J. R. Langmuir 2006, 22, 7279. (22) Wang, L. J.; Zhang, W.; Qiu, S. R.; Zachowicz, W. J.; Guan, X. Y.; Tang, R. K.; Hoyer, J. R.; De Yoreo, J. J.; Nancollas, G. H. J. Cryst. Growth 2006, 291, 160. (23) Bowers, G. M.; Kirkpatrick, R. J. Cryst. Growth Des. 2011, 11, 5188. (24) Freeman, C. L.; Harding, J. H.; Quigley, D.; Rodger, P. M. J. Phys. Chem. C 2011, 115, 8175. (25) Weiner, S.; Addadi, L. Annu. Rev. Mater. Res. 2011, 41, 21. (26) Orme, C. A.; Noy, A.; Wierzbicki, A.; McBride, M. T.; Grantham, M.; Teng, H. H.; Dove, P. M.; DeYoreo, J. J. Nature 2001, 411, 775. (27) Maruyama, M.; Tsukamoto, K.; Sazaki, G.; Nishimura, Y.; Vekilov, P. G. Cryst. Growth Des. 2009, 9, 127. (28) Deganello, S.; Piro, O. E. Neues Jahrb. Mineral. 1981, 81. (29) Cody, A. M.; Horner, H. T.; Cody, R. D. Scanning Electron Microsc. 1982, 1, 13. (30) Millan, A. Cryst. Growth Des. 2001, 1, 245. (31) Webb, M. A. Plant Cell 1999, 11, 11. (32) Wierzbicki, A.; Sikes, C. S.; Madura, J. D.; Stevens, E. D.; Martin, K. L. Calcif. Tissue Int. 1995, 56, 8.

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CONCLUSIONS The findings presented above provide a molecular-level view of how the chirality of impurities and additives is transferred to crystal morphology. As expected, for chiral modification to occur, the existence of mirror symmetry is prerequisite and can either be obtained through the presence of a mirror plane in the crystal structure or the formation of twin planes during crystal nucleation and growth. In addition, there must be a stereochemical match between the step structure and one of the enantiomers. Hydrogen bonding may also play an important role in steering the structural recognition. Previous studies have shown that even single aspartic acid enantiomers can break crystal symmetry through modification of either the thermodynamics26 or kinetics27 of growth through, respectively, the step edge free energy or the solute attachment and detachment rates. We also expect that both factors could be at work in the case of COM; however, because the current study only investigated the impact on step speeds and growth shapes, the observed effects must be related to kinetic factors. Whatever factors dominate, modification of step edge free energies or changes in step kinetics, they all result from the preferential binding of specific chiral molecules to steps related by mirror symmetry. Combined with the results of the previous study of calcium carbonate system, our studies suggest that formation of chiral morphologies in minerals may follow the general rule that molecular chirality of biomolecules is transferred to crystal morphology through preferential binding to mirror symmetryrelated surface steps.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (S.R.Q.); [email protected] (Y.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant DK61673 from the National Institutes of Health, the Lawrence Scholar Program Student Fellowship from Lawrence Livermore National Laboratory, Office of Science, Office of Basic Energy Sciences of the US Department of Energy under Contracts DE-AC52-07NA27344 and DE-AC02-05CH1123. Y.H. acknowledges support from the Office of Naval Research (ONR) (award N00014-08-10985). K.R.C. acknowledges initial support for graduate study 5946

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