Modulation of Crystal Growth by the Terminal Sequences of the

Nov 4, 2008 - ABSTRACT: The formation of calcite in the mollusk shell prismatic layer ... studies indicate that the prismatic-associated protein super...
2 downloads 0 Views 1MB Size
Download PDF
Modulation of Crystal Growth by the Terminal Sequences of the Prismatic-Associated Asprich Protein Katya Delak,§,† Jennifer Giocondi,§,‡ Christine Orme,‡ and John Spencer Evans*,† Laboratory for Chemical Physics, Center for Biomolecular Materials Spectroscopy, New York UniVersity, 345 E. 24th Street, Room 1007, New York, New York 10010, and, Department of Chemistry and Materials Science, Lawrence LiVermore National Laboratory, LiVermore, California 94551

CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 12 4481–4486

ReceiVed April 25, 2008; ReVised Manuscript ReceiVed October 1, 2008

ABSTRACT: The formation of calcite in the mollusk shell prismatic layer requires the participation of various proteins. Recent studies indicate that the prismatic-associated protein superfamily, Asprich, is capable of in vitro stabilization of amorphous calcium carbonate (ACC), a precursor phase of prismatic calcite. To learn more about the molecular behavior of Asprich, we performed experiments on two highly conserved sequences derived from Asprich: Fragment-1, a 48 AA N-terminal cationic-anionic sequence, and Fragment-2, a previously characterized 42 C-terminal AA anionic sequence. SEM analyses reveal that Fragment-1 induces polycrystalline, radial aggregate assemblies of calcite, with evidence of surface porosities. AFM flow cell experiments demonstrate that Fragment-1 is multifunctional and its mineralization behavior is qualitatively similar to that reported for Fragment-2 except for hillock step kinetics. Surprisingly, when Fragment-1 and Fragment-2 are present together within the same assay, we observe phase stabilization of vaterite on Kevlar substrates and amorphous-appearing islands on calcite substrates. We believe that island formation on the calcite substrate results from the deposition of peptide-mineral clusters onto calcite hillock terrace surfaces. These events may also take place on the Kevlar substrate as well, where either vaterite or calcite form. The most significant feature is that a mixture of Fragment-1 + Fragment-2 are required to induce these effects and that the individual sequences themselves do not have this capability. These results indicate that these conserved terminal Asprich sequences jointly exhibit mineralization behavior (i.e., phase stabilization) that is qualitatively similar to the parent protein, and, parallels the in vitro findings reported for other calcite and aragonite - associated polypeptide sequences. It is likely that the sequence features of Asprich may be used to design crystal growth mimetics that can modulate crystal growth within the laboratory setting. Introduction One of the more interesting aspects of the biomineralization process is the participation of specific mineral regulatory proteins.1-4 As an example, in some mollusk shells there exist two layers, nacreous and prismatic, each of which possess different polymorphs of calcium carbonate. It is now becoming clear that mineral formation within the nacreous and prismatic layers of the mollusk shell involves the participation of distinct proteins with different sequence features.3-6 Recent studies of nacre (aragonite) associated polypeptides7-10 reveal that these sequences consist of a mixture of anionic, cationic, polar and hydrophobic sequence regions. Some of these nacre polypeptides behave in a multifunctional capacity in vitro, that is, they simultaneously block and accelerate certain aspects of calcium carbonate crystal growth,10,11 and/or, induce the formation of new mineral deposits.11 Conversely, many prismatic (calcite) associated polypeptide sequences are highly negatively charged and consist of significant percentages of Asp and Glu residues.12-14 Recent studies indicate that prismatic - associated proteins, such as caspartin,15 calprismin,15 and prismalin,16 are capable of modulation calcium carbonate crystal growth and morphology. However, compared to nacre-specific sequences, little is known regarding the mineral modification capabilities of other prismatic-associated sequences. Recently, a subfamily of seven proteins associated with the prismatic layer of the bivalve, Atrina rigida, were identified and sequenced.12 These proteins, designated as Asprich “a” through * To whom correspondence should be addressed. Tel: 2129989605. Fax: 2129954087. E-mail: [email protected]. § Indicates equal contributors to this work. † New York University. ‡ Lawrence Livermore National Laboratory.

“g”, are multidomain in nature and consist of C- and N-terminal sequences that are highly conserved witin this superfamily. Recent studies indicate that recombinant Asprich induces and stabilizes amorphous calcium carbonate (ACC) in vitro.12b Hence, Asprich sequences possess important information with regard to essential features that enable phase stabilization and transformation to prismatic calcite. To obtain this sequence information, a reductionist approach has been employed to study these highly conserved Asprich N- and C-terminal sequences as individual entities. The rationale for studying protein sequence fragments and employing them individually and in a combinatorial fashion are to learn more about mollusk shell sequences and their effects on mineralization under reproducible conditions. This provides two benefits. First, the molecular behavior of the individual sequences under controlled conditions can provide clues as to their potential participation and function within the parent protein and how this contrasts with sequences obtained from other mollusk shell proteins. Second, the molecular characteristics of these sequences can serve as models for developing proteins or polymers which control or modulate crystal growth for materials or nanotechnological applications. As an example, the conserved C-terminal 42 amino acid (AA) Asprich sequence (Fragment-2)12,17,18 generates irregular crystal growth patterns on calcite crystals17 and forms deposits which adsorb onto calcite dislocation hillock terraces, resulting in morphological “sculpting” of calcite into a rounded geometry.18 The mineralization activity of Fragment-2 differs from that observed for nacre-specific protein sequences,11 and this demonstrates that each shell layer employs unique sequence-specific mechanisms to construct distinct mineralized layers. In contrast, there is no information available for the Nterminal Asprich sequence region. This sequence is comprised

10.1021/cg8004294 CCC: $40.75  2008 American Chemical Society Published on Web 11/04/2008

4482 Crystal Growth & Design, Vol. 8, No. 12, 2008

Figure 1. Primary sequence of the conserved N-terminal region of Asprich (Fragment-1). Numbering system (black) corresponds to the cleaved, mature polypeptide, minus the N-terminal signal peptide region.15 Original numbering system12 is shown above Fragment-1 in parentheses. Fragment-2 domain of Asprich is shown for comparison.

of a cationic 7 AA region and a 100 AA Asp, Glu-rich anionic region.12 Additionally, we do not know if both terminal sequences can jointly modulate crystal growth in a manner that mirrors the activity of the Asprich protein. This Report details initial structure-function studies of this N-terminal sequence, and, reports an interesting finding with regard to the potential synergy between the N- and C-terminal sequences of Asprich during in vitro crystal growth. As a starting point, we synthesized a 48 AA synthetic polypeptide (Fragment-1) representing a chemically synthesizable portion of the N-terminal portion of Asprich (Figure 1).12 Using SEM, we find that Fragment-1 induces the formation of radial polycrystalline clusters of calcite that feature surface voids/porosities, similar to what has been previously reported for egg shell calcite-associated19-22 and prismatic-associated15 proteins. AFM flow cell studies reveal that Fragment-1 is multifunctional and is qualitatively similar to Fragment-2 in terms of hillock morphology effects, deposit formation, and crystal interaction, but diverges from Fragment-2 in terms of hillock growth kinetics. Interestingly, a mixture of Fragment-1 and Fragment-2 induces vaterite formation23,24 in our SEM assay systems and round terrace islands in our AFM assay systems, indicating that the integral terminal sequences of Asprich, like the parent protein itself, are jointly capable of phase stabilization. Materials and Methods Polypeptide Synthesis. Free N-terminal, C-R-amide-capped 48-AA Fragment-1 polypeptide was synthesized and purified at the 100 micromole level at the Wm. Keck Biotechnology Peptide Synthesis Facility, Yale University, by Dr. Janet Crawford, using protocols described in our earlier work for other Asprich sequences.17,18 The N-acetyl-capped, free alpha-carboxyl 42 AA Fragment-2 polypeptide was synthesized and purified as previously described.17,18 C- and N-Terminal alpha amide capping procedures were performed to simulate peptide bond attachment.7,17,18 After resin cleavage and reverse-phase HPLC purification (Waters C-18 column, >95% pure),17,18 the experimental molecular mass of Fragment-1 was determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to be 5178.1 Da, in agreement with the theoretical value of 5177.2 Da. In Vitro Kevlar Crystal Growth Assays. We employed a polyimide (Kevlar) assay17 for the nucleation of calcium carbonate crystals in the presence of Fragments-1, -2, and a 1:1 mol ratio mixture of Fragment-1 + Fragment-2. Total polypeptide assay concentrations were 1 × 10-5, 5 × 10-5, and 1 × 10-4 M. Negative control conditions consisted of no added peptide. Assay conditions and sample workup for SEM imaging were conducted as described in our previous Asprich

Delak et al. C-terminal polypeptide studies.17,18 Scanning electron microscopy imaging was conducted using a Hitachi S-3500N SEM microscope at 5 kV after thin Au coating of samples. The SEM images presented in this report are representative of 10-20 different crystals in each assay sample. Cropping of SEM images and adjustment of brightness/darkness and contrast levels were performed using Adobe Photoshop. Micro-Raman Spectroscopy of Kevlar-Nucleated Crystals. To verify the formation of calcium carbonate polymorphs in the presence of Fragment-1 + Fragment-2 mixture, micro-Raman analysis was performed on Kevlar-nucleated crystals which exhibited both rhombohedral and nonrhombohedral features, and, for comparison, the Kevlar threads themselves. Micro-Raman spectroscopy measurements were made with a commercial instrument (Horiba-Jobin Yvon, Edison, NJ) employing a 10 mW 632.8 nm He-Ne laser as the probe beam. The instrument settings were as follows: 600 spacing/mm grating, 300 micron hole, 100 micron slit, and 20× or 100× microscope objectives were used. Each spectrum was obtained by averaging three 60 s runs. In Situ AFM. AFM experiments were performed on surfaces of anchored calcite crystals (freshly cleaved geologic calcite from Brazil, e1 mm each dimension) that were subjected to overgrowth via exposure to a supersaturated solution of CaCl2/NaHCO3 (2.5 mM CaCl2, 2.5 mM NaHCO3 in deionized distilled water) and imaged in real time using an atomic force microscope outfitted with a commercially available fluid cell (Nanoscope III, Digital Instruments, Santa Barbara, CA).11,18 Prior to peptide introduction into the fluid cell system, each calcite crystal was equilibrated via exposure to supersaturated solution; the choice of flow rate (1 mL/min) was such that step kinetics were not limited by bulk diffusion. Once well-defined hillocks were detected, polypeptides were then introduced using a freshly prepared supersaturated solution at the same flowrate. This solution consisted of a given peptide dissolved in NaHCO3 solution, which was filtered using a 0.2 µM PVDF Gelman Acrodisc11,18 and then mixed with CaCl2 solution, with the final concentration of both CaCl2 and NaHCO3 ) 2.5 mM and the final peptide concentration ) 3, 6, or 12 µM. We also ran parallel imaging experiments on a 1:1 peptide mixture of 3 µM Fragment-1 + 3 µM Fragment-2. Negative control conditions utilized supersaturated solutions that were devoid of any added peptide; these solutions normally have a pH of 8.3, and therefore were adjusted to 8.1 by adding a minute amount of dilute aqueous HCl. The addition of Fragment-1 to the unadjusted negative control solution (pH ) 8.3) led to an approximate downshift to pH 7.3, which was then adjusted to pH 8.1 with minute volumes of KOH to avoid downshift in supersaturation or slowing of step growth kinetics. The supersaturation ratio S ) [{Ca2+}{CO32-}/Ksp,calcite] and the activity ratio {Ca2+}/(CO32-} of pure solutions (no polypeptide) at pH 8.1 were computed using Geochemists’ Workbench software (v.5, Rockware, Inc.) and found to be 6.1 and 130, respectively. The chelation of calcium ions by the peptide is not expected to shift the supersaturation appreciably due to the low peptide/Ca(II) ratio. All imaging was performed on the {104} cleavage plane of calcite. AFM fluid cell imaging was performed at room temperature with image collection commencing 5 min after the introduction of each polypeptide.11,18 In situ images were collected in contact mode using Si3N4 tips and were limited to regions undergoing step growth at dislocation hillocks of calcite. The imaging force was reduced to the minimum possible value that allowed the tip to remain in contact with the surface, such that there was no measurable effect on the growth kinetics.11,18 AFM imaging was typically performed once with a given polypeptide sample, and no local erosion or local enhancement of step velocities was observed due to tip effects. Note that step-angle distortion exists in the images because the step front advances during the scan time. Images reported here are not corrected for this effect.11,18 All images were processed with Image SXM (version 174-1X) for color, brightness, contrast adjustment, and examination of height profile. Dry imaging of calcite crystals exposed to Fragment-1 were obtained by first removing the crystal from the fluid cell, rinsing the crystal with 10 mL of deionized distilled water and drying with clean compressed nitrogen gas. Samples were then imaged in tapping mode. Final image adjustment for all samples was made using Adobe Photoshop. In addition to the AFM studies described above, we conducted parallel AFM imaging studies of calcite dislocation hillock growth in the presence of a 1:1 peptide mixture composed of 50 µM Fragment-1 + 50 µM Fragment-2, over a total period of 18 h. Note that these higher concentration studies were performed with some modifications to provide a comparison with our Kevlar nucleation assays described in

Crystal Growth Modulation by Asprich Protein

Figure 2. Scanning electron microscopy images of in vitro Kevlar calcium carbonate assay systems. (A) negative control assay, which features typical rhombohedral calcite crystals; (B) Fragment-1, 50 µM. Here, arrows denote gaps between crystals, presumable arising from crystal twinning; (C) Fragment-1, 100 µM; (D) as per (C), but at higher magnification, revealing the presence of surface porosities (arrows). Scalebars indicate image dimensions. the preceding sections. A 10 mL solution was used to flow through the imaging flow cell at a slower rate (e0.5 mL/min) with recirculation of the solution to extend the experiment to longer time intervals that matched those of the Kevlar assay study (i.e., 18 h). Thus, these longterm studies are expected to experience concentration and pH variations over time; in fact, the final pH of the solution at the conclusion of the experiment was found to be ∼8.9. CD Spectrometry. Circular dichroism (CD) spectra were obtained for Fragment-1 and Fragment-2 at 20 °C, using an AVIV 60 CD spectrometer (60DS software version 4.1t). The CD spectrometer was previously calibrated with d-10-camphorsulfonic acid. The peptide samples (apo form) were dissolved and diluted to final concentrations of 8 µM in 100 µM Tris HCl (pH 7.5). For all spectra, wavelength scans were conducted from 185 to 260 nm with appropriate background buffer and CaCl2 subtraction, using a total of four scans with 1 nm bandwidth and 0.5 nm/s scan rate17,18 In all CD spectra, mean residue ellipticity [θM] is expressed in deg · cm2 · dmol-1.17,18

Results and Discussion Fragment-1 Modulation of Calcite Crystal Morphology and Kinetics. Fragment-1 induces the growth of micron-sized spherical assemblies or clustered arrays of polycrystalline calcite clusters on Kevlar threads (Figure 2). We also note the presence of voids or porosities on the surface of calcite crystals grown in the presence of Fragment-1 (Figure 2D, note arrows). Note that these surface features have been observed in crystal growth assays containing egg shell-associated polypeptides.19-22 This phenomenon was also reported for Asprich Fragment-2,17 and we believe that these surface voids arise from peptide-mineral interactions that lead to uneven crystal growth rates and “fingering” effects,17 as we note below in our AFM studies. Interestingly, the spherical or radial assemblies observed in Figure 2 are composed of small rhombohedral crystals that feature gaps or spacings between crystals. There may be one or more underlying mechanisms which would explain the formation of crystal assemblies with these features. For example, Fragment-1-induced crystal twinning may be occurring (Figure 2B,C), as evidenced by the presence of gaps or spaces between adjacent calcite rhombohedral crystals (note arrows in Figure 2B).15 Note that the biological twinning phenomenon has been reported for calcite crystals growth in the presence of prismatic-

Crystal Growth & Design, Vol. 8, No. 12, 2008 4483

Figure 3. AFM contact mode images of the dislocation hillock region of calcite crystals. (A) Fragment-1, 6 µM; (B) Fragment-1, 12 µM; (C) dry topographic image of hillock terrace region after exposure to 6 µM Fragment-1. All images are shown in the same orientation with the two acute steps at the top of the images. AFM inset images are those recorded immediately prior to the introduction of Fragment-1 (i.e., negative control conditions). Scalebars indicate image dimensions. Atomic force microscopy on the {104} cleavage plane of calcite reveals that dislocation hillock growth proceeds via atomic step advancements over the range of conditions used in these experiments. The atomic step directions reflect typical rhombohedral calcite crystal morphology with step risers along four of the six crystallographically equivalent {104} facets (A). Two of the steps are typically termed “acute” (top portion of A) due to the acute angle that the step riser makes with respect to the cleavage plane, and two of the steps are termed “obtuse” (bottom portion of A) due to the obtuse angle the step riser makes with respect to the cleavage plane.11,18

associated proteins.15b However, since X-ray diffraction techniques have not been applied to the crystals in our assays, there is no physical evidence to verify crystal twinning. Alternatively, other events may have led to the formation of the gaps, spacings, or radial orientations of the aggregate crystals, such as a gradual shift in crystal orientation across the aggregate in response to shrinkage stresses induced by ACC transformation.12,24 Although the true mechanism of crystal aggregate formation is not known, the fact that Fragment-1 generates these clusters indicates that this sequence is controlling mineralization events either at the mineral interface or in solution, ultimately affecting calcite crystal morphology in this unique fashion. Using AFM imaging, we note that the introduction of Fragment-1 produces several significant effects on calcite dislocation hillock morphology that are shared with those produced by Fragment-2 (Figure 3).18 First, we note that Fragment-1, like Fragment-2, induces the formation of clusters on terrace surfaces, and we believe that these clusters represent peptide-mineral aggregates or deposits.11,18 Interestingly, at low polypeptide concentrations, steps continue to grow under these clusters. Second, the morphology of acute-obtuse corner sites becomes rounded, losing the sharp delineation between acute andobtusestepdirections,leadingtoanoverallovalmorphology.11,18 Third, we observe acute step “bunching” as noted by the appearance of roughened acute step edges that are not observed in the negative control scenario. Fourth, we observe that the

4484 Crystal Growth & Design, Vol. 8, No. 12, 2008

Delak et al.

Figure 4. Far UV CD spectra of Fragment-1, Fragment-2 at pH 7.5 in the presence of 100 µM Tris-HCl buffer.

obtuse steps become very rough over time with a “finger” geometry that can be distinguished from classically scalloped pinning morphologies in the following way. This finger morphology (and therefore the step kinetics) is anisotropic with long, narrow peninsulas growing through blocked regions. This anisotropy is not typical of classical step-pinning models although curvature controlled effects may also occur. The appearance of a finger is consistent with limited kink mobility along the step so that growth occurs via local attachment at regions that are not blocked by impurities. The sides of these peninsulas grow slowly or not at all so that they maintain a high aspect ratio. Interestingly, finger regions have also been observed in AFM experiments with Fragment-2, where the fingering breaks up the steps, creating a porous surface (see for example Figure 3C).18 It is presumed that the porosities observed on macroscopic calcite crystals (Figure 2D) arise from this fingering process. Kinetic measurements (Supporting Information, Figure S1) indicate that obtuse step velocities are higher in the presence of Fragment-1 (as compared to peptide-free solutions) and remain relatively constant when plotted against step distance from the dislocation source. In contrast, earlier experiments conducted with the Asprich Fragment-2 indicated that this sequence induces initial velocity acceleration followed by deceleration due to pinning.18 In conclusion, although the mineral recognition properties, deposit formation, finger formation and induced hillock morphologies of Fragment-1 and Fragment-2 are qualitatively similar, we find that both sequences differ in terms of hillock growth kinetics. We believe that this difference in kinetics reflects the different modes that each sequence utilizes to bind and pin obtuse steps. Structural features of Fragment-1. The conformation of Fragment-1 was qualitatively investigated using CD spectrometry (Figure 4) and compared against data previously obtained for Fragment-2.17,18 Here, we note that apo-Fragment-1 is qualitatitively similar to Fragment-2: both sequences exhibit a major (-) ellipticity band (π-π* transition) at 198 nm, corresponding to random coil or unstructured conformation.11,17,18 However, Fragment-1 also possesses a minor, (+) band (n-π* transition) centered near 218 nm, corresponding to polyproline Type II (PPII) extended helical conformation.17,18 Given that the 7 AA cationic region of Fragment-1 possesses a Pro residue and three positively charged residues (Figure 1), we infer that the PPII-like characteristics of Fragment-1 may arise from these compositional features.25 Thus, although Fragment-2 and Fragment-1 are qualitatively similar in structure, one structural trait is unique to Fragment-1, and that is the presence of PPII

Figure 5. Visual and spectroscopic analyses of crystal growth in the presence of Fragment-1 and Fragment-2. (A) SEM image of calcite crystals obtained from in vitro Kevlar calcium carbonate negative control assay; (B) SEM image of calcium carbonate crystals grown in the presence of 1:1 mol mixture (total peptide concentration ) 100 µM) of Fragment-1 + Fragment-2. Scalebar ) 10 microns. Note vaterite-type morphology. (C) and (D) are light microscope images of a rhombohedral calcite (C) and vaterite-type crystals (D) obtained from 1:1 mixture assay and utilized for microRaman analysis. (E) Raman analysis of “C” and “D” crystals along with the experimental spectra of the Kevlar fibers and the literature values from reference for calcite (blue) and vaterite (red), shown as bars along the bottom of this graph for comparison. The vertical axis is intensity in arbitary units. The Raman modes for both calcite and vaterite can be found in Table S1 (Supporting Information).

structure. At this time we do not know what region(s) of either sequence contain PPII structure or how this structure plays a role in the in vitro mineralization events noted in our report. Crystal Growth Behavior in the Presence of Fragment-1 and Fragment-2 Polypeptides. In our previous studies, it was noted that a 1:1 mixture of the two subdomains that comprise Fragment-2 alters crystal growth in a manner that differs from the mineralization effects noted for Fragment-2 and for the individual subdomains themselves.17,18 To extend this observation further, we ran parallel Kevlar mineralization assays using a 1:1 mol ratio of Fragment-1 and Fragment-2 (Figure 5). Here, we note that the formation of rhombohedral calcite (Figure 5A,C) is also accompanied by the formation of spherulitic clusters that resemble vaterite (representing approximately 10-30% of the total crystals formed, Figure 5B,D). Note that the formation of vaterite was not observed in the presence of Fragment-1 alone (Figure 2) or in our previous studies with Fragment-2 alone.18 Given the small sizes and limited numbers of crystals grown in our Kevlar assays, the use of bulk characterization techniques, such as X-ray diffraction, are seriously limited for distinguishing different polymorph structures. However, techniques such as micro-Raman spectroscopy can be employed to focus on

Crystal Growth Modulation by Asprich Protein

Figure 6. AFM contact mode images of the dislocation hillock region of calcite crystals in the presence of 1:1 mol mixture of Fragment-1 + Fragment-2. Image (A) represents the negative control assay; Images (B), (C), and (D) are taken in the presence of 3 µM Fragment-1 + 3 µM Fragment-2 at t ) 20, 199, and 211 min from the time of peptide addition, respectively. Image dimensions: A, B, C ) 15 × 15 µm, D ) 8 × 8 µm.

individual crystals and verify their lattice structures. Using micro-Raman spectroscopy, we confirmed that the spherulitic clusters are vaterite (Figure 5E) via comparison to spectra obtained for calcium carbonate polymorphs (Supporting Information, Table S1). The two key areas of comparison are the lattice modes at low wavenumbers and symmetric stretch (η1) mode around 1085 cm-1. For vaterite, there is a triplet in the lattice mode region and a split peak in the symmetric stretch region that distinguishes it from both calcite and aragonite. As expected, the negative control crystal exhibits the typical rhombohedral shape of calcite and the micro-Raman spectrum confirms this assignment (Figure 5). We did not detect the presence of ACC in these assays; however, given that Fragment 1 generates radial crystal aggregates (Figure 2), and, that spherulitic vaterite crystals are forming in our assays (Figure 5), it is possible that an amorphous calcium carbonate phase was initially present prior to the appearance of calcite or vaterite. To further investigate this interesting finding, we utilized AFM to monitor calcite hillock growth and kinetics in the presence of a 1:1 Fragment-1/Fragment-2 mixture (Figure 6) as a function of time. After 20 min of exposure, we observe hillock features that are associated with the presence of either Fragment-1 or Fragment-2: the formation of clusters on terrace surfaces, the roundening of acute-obtuse corner regions and the fingering of the obtuse steps that leads to porosity formation. Simulatenously, a new feature begins to emerge, namely, the formation of two-dimensional round islands on hillock surfaces. As time evolves, the predominant growth mechanism changes from step growth to nucleation and spreading of twodimensional islands and the formation of clusters on hillock surfaces (Figure 6). Hence, the 1:1 mixture leads to the induction of morphological features and crystal growth mechanisms that are atypical for the individual fragments. We repeated these AFM flowcell experiments at higher peptide concentrations (i.e., 100 µM, Supporting Information,

Crystal Growth & Design, Vol. 8, No. 12, 2008 4485

Figure S2) utilized for the Kevlar assays. Under these conditions, we observe two competing processes taking place on the teraces: the formation of etch pits and the simultaenous nucleation of two-dimensional round islands. Due to a several minute gap in imaging after new solutions were introduced into the fluid cell, we cannot be certain that the pits represent the active dissolution of calcite, or, peptide induced “dead” spots on the crystal that material grows around thereby creating a hole. Nonetheless it is clear that the growth mode has changed and that the new growth does not have facets and that pit formation ceases after the new overlayer has grown. Using Raman spectroscopy, we were unable to detect the presence of noncalcite calcium carbonate polymorphs in this new overgrowth layer (data not shown). Given that the forming layer may not be sufficiently thick to be detected against the larger calcite background, or, that assay solution conditions may have led to calcite transformation over time, we cannot identify the true mineral phase of these islands. We believe that island formation on the calcite substrate results from the deposition of peptide-mineral clusters onto terrace surfaces, which are observed in our AFM experiments with individual Fragment-218 and Fragment-1 (Figure 3) peptides. Once these deposits are on the terrace surfaces, further nucleation events occur at these regions, possibly involving the transient formation of ACC, since this would be consistent with the rounded island appearance. This, in turn, rapidly crystallizes into calcite on the calcite substrate (Figure 6). These events may also take place on the Kevlar substrate as well (Figure 5), where either vaterite or calcite form. Regardless of the substrate, the most significant feature is that a mixture of Fragment-1 + Fragment-2 are required to induce these effects (Figures 5 and 6), and that the individual sequences themselves do not have this capability. In summary, the N- and C-terminal domains of the Asprich protein superfamily are multifunctional and can induce cluster deposits on calcite terraces, pin steps, and affect calcite hillock morphology or shape in similar ways (Figure 3). In addition, these two conserved sequence domains functionally diverge in a number of important areas. First, only Fragment-1 can induce polycrystalline aggregation (Figure 2). The fact that crystal aggregates form with radial orientations and with gaps or spaces between crystals suggests a number of possible scenarios, such as twinning,15 or shifts in crystal orientation or shrinkage stresses arising from the transformation of ACC12,24 at some point during the assay period. These issues will be followed up in a subsequent report. Second, each sequence exhibits slightly different propensities for obtuse step-pinning which causes differences in the step growth kinetics (Supporting Information18 What molecular feature(s) could give rise to these molecular differences? One obvious starting point would be the unique 7 AA cationic region within the Fragment-1 sequence. Here, it is likely that the presence of Pro and a short polyelectrolyte cluster region induce some degree of PPII structure25 that we detect in our CD experiments (Figure 4A). We believe that this cationic region influences Fragment-1 to form clusters or deposits and/ or interact with specific calcite hillock features (Figure 3) which, in turn, might influence morphology in a manner that is distinct from that of Fragment-2. Another sequence feature which distinguishes Fragment-1 from Fragment-2 is the Asp and Glu residue content and the corresponding net electrostatic charge. In Fragment-1, 31% of the sequence is Asp, 13% of the sequence is Glu, and the ratio of Asp/Glu ) 2.5; the net charge of this sequence ) -17 at neutral pH.26 In Fragment-2, 36% of the sequence is Asp, 20% of the sequence is Glu, the Asp/

4486 Crystal Growth & Design, Vol. 8, No. 12, 2008

Glu ratio ) 1.9, and the net charge ) -24.26 Hence, amino acid composition, backbone structure, and electrostatics may jointly contribute to the molecular differences that we observe in vitro. The most striking finding of our study was the stabilization of vaterite (Figure 5) and formation of amorphous or roundappearing islands (Figure 6) on calcite substrates in the presence of Fragment-1 and Fragment-2. These findings suggest that Fragment-1 and Fragment-2 jointly exhibit phase stabilization properties, and we note that phase stabilization (i.e., ACC) is a reported feature of the parent protein, Asprich.12 As a consequence, we believe that the in vitro mineralization behavior of Fragment-1 and Fragment-2 is quallitatively similar to that of Asprich itself. Although the true identity of calcium carbonate phase(s) within these islands is unknown (Figure 6), their round appearance and the fact that Fragment-1 and Fragment-2 individually induce amorphous deposits upon terrace surfaces (Figure 3)18 suggests that at some point ACC formation or nucleation occurs on hillock surfaces. At present, we do not know the mechanism(s) by which Fragment-1 + Fragment-2 stabilize these phases, nor do we know what percentage of either fragment sequence interacts or participates in the formation of noncalcitic phases within our assays. However, it has been suggested that protein stabilization of spherulitic vaterite or ACC arises from polypeptide carboxylate interactions with the mineral phase.12,23 If true, then it is likely that the high carboxylate Asp, Glu content in both Asprich terminal sequences exerts some effect on polypeptide-mineral, polypeptide-Ca (II), or even polypeptide-polypeptide complexation and assembly. In turn, one or more of these molecular phenomena facilitate phase stabilization. Obviously, additional studies will be required to explore these potential mechanisms and synergistic interactions between Fragment-1 and Fragment-2 and compare these to what takes place within the Asprich protein itself. The fact that anionic protein sequences can stabilize phases such as vaterite or ACC may prove to be a useful model for developing polypeptidebased phase transformation and crystal engineering techniques in the laboratory for materials and nanotechnology applications. Acknowledgment. This work was supported by funding from the National Science Foundation (DMR-0704148, to J.S.E.). Portions of this work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. This paper represents contribution number 46 from the Laboratory for Chemical Physics, New York University. Supporting Information Available: Step velocity plot (Figure S1), AFM imaging of hillock regions in the presence of 100 µM peptide concentrations (Figure S2), and table of Raman wavenumbers and modes for calcium carbonates (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989; pp 1-50.

Delak et al. (2) Levi-Kalisman, Y.; Falini, G.; Addadi, L.; Weiner, S. J. Struct. Biol. 2001, 10, 4372–4337. (3) Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689–702. (4) Fritz, M.; Belcher, A. M.; Radmacher, M.; Walters, D. A.; Hansma, P. K.; Stucky, G. D.; Morse, D. E.; Mann, S. Nature 1994, 371, 49– 51. (5) Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Nature 1996, 381, 56–58. (6) Thompson, J. B.; Paloczi, G. T.; Kindt, J. H.; Michenfelder, M.; Smith, B. L.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Biophys. J. 2000, 79, 3307–3312. (7) Michenfelder, M.; Fu, G.; Lawrence, C.; Weaver, J. C.; Wustman, B. A.; Taranto, L.; Evans, J. S.; Morse, D. E. Biopolymers 2003, 70, 522–533. Erratum, Biopolymers 2004, 73, 299. (8) Samata, T.; Hayashi, N.; Kono, M.; Hasegawa, K.; Horita, C.; Akera, S. FEBS Lett. 1999, 462, 225–229. (9) Miyashita, T.; Takagi, R.; Okushima, M.; Nakano, S.; Miyamoto, H.; Nishikawa, E.; Matsushiro, A. Mar. Biotechnol. 2000, 2, 409–418. (10) Fu, G. S.; Qiu, R.; Orme, C. A.; Morse, D. E.; De Yoreo, J. J. AdV. Mater. 2005, 17, 2678–2683. (11) (a) Kim, I. W.; Darragh, M.; Orme, C. A.; Evans, J. S. Cryst. Growth Des. 2006, 6, 6–10. (b) Walters, D. A.; Smith, B. L.; Belcher, A. M.; Paloczi, G. T.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Biophys. J. 1997, 72, 1425–1433. (12) (a) Gotliv, B.-A.; Kessler, N.; Sumerel, J. L.; Morse, D. E.; Tuross, N.; Addadi, L.; Weiner, S. Chem. Bio. Chem. 2005, 6, 304–314. (b) Politi, Y.; Mahamid, J.; Goldberg, H.; Weiner, S.; Addadi, L. CrystEngComm 2007, 9, 1171–1177. (13) Tsukamoto, D.; Sarashina, I.; Endo, K. Biochem. Biophys. Res. Commun. 2004, 320, 1175–1180. (14) Sarashina, I.; Endo, K. Am. Mineral. 1998, 83, 1510–1515. (15) (a) Marin, F.; Arnons, R.; Guichard, N.; Stigter, M.; Hecker, A.; Luquet, G.; Layrolle, P.; Alcaraz, G.; Riondet, C.; Westbroek, P. J. Biol. Chem. 2005, 280, 33895–33908. (b) Pokroy, B.; Kapon, M.; Marin, F.; Adir, N.; Zolotoyabko, E. Proc. Natl. Acad. Sci., U. S. A. 2007, 104, 7337–7341. (16) Suzuki, M.; Murayama, E.; Inoue, H.; Ozaki, N.; Tohse, H.; Kogure, T.; Nagasawa, H. Biochem. J. 2004, 382, 205–213. (17) Collino, S.; Kim, I. W.; Evans, J. S. Cryst. Growth Des. 2006, 6, 839– 842. (18) Kim, I. W.; Giocondi, J.; Orme, C.; Collino, S.; Evans, J. S. Cryst. Growth Des. 2008, 8, 1154–1160. (19) Ajikumar, P. K.; Lakshminarayanan, R.; Ong, B. T.; Valiyaveettil, S.; Kini, R. M. Biomacromolecules 2003, 4, 1321–1326. (20) Lakshminarayanan, R.; Valiyaveettil, S.; Rao, V. S.; Kini, R. M. J. Biol. Chem. 2003, 278, 2928–2936. (21) Ajikumar, P. K.; Lakshminarayanan, R.; Ong, B. T.; Valiyaveettil, S.; Kini, R. M. Biomacromolecules 2003, 4, 1321–1326. (22) Reyes-Grajeda, J. P.; Moreno, A.; Romero, A. J. Biol. Chem. 2004, 279, 40876–40881. (23) Lakshminarayanan, R.; Chi-Jin, E. O.; Loh, X. J.; Kini, R. M.; Valiyaveettil, S. Biomacromolecules 2005, 6, 1429–1437. (24) Pokroy, B.; Zolotoyabko, E.; Adir, E. Biomacromolecules 2006, 7, 550–556. (25) (a) Cubelis, M. V.; Caillez, F.; Blundell, T. L.; Lovell, S. C. Proteins: Struct. Funct. Bioinform. 2005, 58, 880–892. (b) Kentsis, A.; Mezei, M.; Gindin, T.; Osman, R. Proteins: Struct. Funct. Bioinform. 2004, 55, 493–501. (c) Chellgren, B. W.; Creamer, T. P. Biochemistry 2004, 43, 5864–5869. (d) Eker, F.; Griebenow, K.; Cao, X.; Nafie, L. A.; Schweitzer-Stenner, R. Biochemistry 2004, 43, 613–621. (26) These charge calculations take into account the presence of C-terminal amide capping, and, the Arg, Lys charge contributions arising from the 7 AA catioinic sequence of Fragment-1.

CG8004294