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Intrinsically Disordered Mollusk Shell Prismatic Protein That Modulates Calcium Carbonate Crystal Growth Moise Ndao,† Ellen Keene,‡ Fairland F. Amos,† Gita Rewari,† Christopher B. Ponce,† Lara Estroff,‡ and John Spencer Evans*,† Laboratory for Chemical Physics, New York University, 345 East 24th Street, New York, New York 10012, and Department of Materials Science and Engineering, Cornell University, Ithaca, New York 14853 Received March 18, 2010; Revised Manuscript Received August 27, 2010

The formation of calcite prism architecture in the prismatic layer of the mollusk shell involves the participation of a number of different proteins. One protein family, Asprich, has been identified as a participant in amorphous calcium carbonate stabilization and calcite architecture in the prismatic layer of the mollusk, Atrina rigida. However, the functional role(s) of this protein family are not fully understood due to the fact that insufficient quantities of these proteins are available for experimentation. To overcome this problem, we employed stepwise solid-phase synthesis to recreate one of the 10 members of the Asprich family, the 61 AA single chain protein, Asprich “3”. We find that the Asprich “3” protein inhibits the formation of rhombohedral calcite crystals and induces the formation of round calcium carbonate deposits in vitro that contain calcite and amorphous calcium carbonate (ACC). This mineralization behavior does not occur under control conditions, and the formation of ACC and calcite is similar to that reported for the recombinant form of the Asprich “g” protein. Circular dichroism studies reveal that Asprich “3” is an intrinsically disordered protein, predominantly random coil (66%), with 20-30% β-strand content, a small percentage of β-turn, and little if any R-helical content. This protein is not extrinsically stabilized by Ca(II) ions but can be stabilized by 2,2,2-trifluoroethanol to form a structure consisting of turn-like and random coil characteristics. This finding suggests that Asprich “3” may require other extrinsic interactions (i.e., with mineral or ionic clusters or other macromolecules) to achieve folding. In conclusion, Asprich “3” possesses in vitro functional and structural qualities that are similar to other reported for other Asprich protein sequences.

Introduction The mollusk shell is a true biocomposite that, in some species, is comprised of two physically distinct layers of calcium carbonate that coexist with biomacromolecules.1-9 The prismatic or calcite layer of the shell is mechanically and structurally distinct from the neighboring fracture-resistant nacre (aragonite) layer.1-10 The prismatic layer consists of long, parallel assemblies of prismatic columns of calcite (single crystals, 100-200 µm in cross-section).1-3 The prismatic layer has a lower fracture toughness compared to the nacreous layer and is more brittle.1-3 Under indentation forces, the prismatic layer experiences radial crack propagation, which serves as a protective shield that resists piercing from predators (i.e., punctureresistance).5,6 Under compressive forces, fracture does occur alongside prismatic crystals and does not completely propagate through the prismatic layer (i.e., crack propagation resistance).5,6 Thus, the calcitic prismatic layer offers an interesting route for defeating catastrophic material failure of the shell, and represents an important biological model system for materials science. The material and biological properties of the mollusk shell prismatic layer have been linked to the presence of specific matrix proteins.10-17 Several protein classes, including silk-like proteins11,12 and acidic proteins13-17 have been uncovered as components within this mineralized tissue. Although the roles of these protein families are not fully understood at present, there is evidence that the silk-like and the acidic proteins perform * To whom correspondence should be addressed. Tel.: 212 998 9605. Fax: 212 995 4087. E-mail: [email protected]. † New York University. ‡ Cornell University.

different roles in the prismatic calcite formation scheme.11-17 As an example, a group of 10 polyacidic proteins, Asprich (“a”-“g”; “1”-“3”), have been identified in the prismatic shell layer of the mollusk, Atrina rigida.15 The Asprich proteins are multidomain in nature and are localized within the calcite prisms and are therefore thought to play a significant role in their formation.15-17 Biophysical studies indicate that the 10 member Asprich family are intrinsically disordered proteins (IDP), meaning that they are largely unfolded and possess functional activity in the unfolded state.18 The highly conserved, unfolded N- and C-terminal domains18,19 of the “a” through “g” protein series can jointly promote the formation of both amorphous calcium carbonate (ACC) and vaterite in vitro.18 These results correlate with in vitro studies of Asprich “g”.16 Thus, Asprich, a member of the intrinsically disordered protein20-22 family, participates in the prismatic crystal building process,17 and may utilize ACC formation and transformation pathways23-26 to control prismatic crystal growth. However, more comprehensive studies of the Asprich protein family have been hampered by the unavailability of these proteins in sufficient quantity for extensive biochemical studies. To date, only the recombinant form of the “g” member of this family has been studied to any significant extent.15-17 One way to circumvent this problem is to use solid-phase chemical synthesis to recreate small native biomineralization proteins (96%, 66 kDa, Sigma-Aldrich) at a concentration of 355 µg/mL, a concentration consistent with other reported mineralization studies.31 In the bottom of each assay well we placed silicon wafer fragments (“P” type [1 0 0], 20 Ohm · cm, 250-350 µm, Silicon Quest Intl., Santa Clara, CA) prior to the start of the assay. These materials were placed such that mineral deposits that settle to the bottom of the dish would be captured on top of these supports by the end of the assay period.28,32 The wafers were then removed and washed using the protocol described in our earlier studies, then allowed to air-dry.28,32 Assay conditions and sample preparation for SEM imaging were conducted as described in our previous studies.19,27,28,32 SEM imaging was conducted using a Hitachi S-3500N SEM microscope at 5 kV. Cropping of SEM images and adjustment of brightness and contrast levels were performed using Adobe Photoshop. Physical Analysis of AsprichsAssociated Mineral Deposits. Si wafers containing washed and dried precipitated assay deposits were analyzed using microRaman spectroscopy and powder X-ray diffraction. For microRaman, spectra for visualized deposits were obtained using a Renishaw InVia Raman Microscope (785 nm laser, 10% power) at Cornell University. The instrument settings were as follows: 1200 spacing/mm grating, 300 µm hole, 100 µm slit, and 50× microscope objectives were used. Powder X-ray diffraction data were collected using a Bruker D8 DISCOVER GADDS Microdiffractometer equipped with a VANTEC-2000 area detector in a φ rotation method. The X-ray generated from a sealed copper tube is monochromated by a graphite crystal and collimated by a 0.5 mm MONOCAP (λ Cu-KR ) 1.54178 Å). The sample detector distance is 150 mm. Two runs with θ1 ) θ2 ) 15 and 30° are collected for each specimen and the exposure time is 600 s per run. Data were merged and integrated by the XRD2EVAL program in the Bruker PILOT Software. The raw file was converted to UXD format by the DIFFRACplusFileExchange, which was later analyzed by the WINPLOTR program. Circular Dichroism Spectroscopy. CD spectra of Asprich “3” in aqueous media, in the presence of CaCl2 and in the presence of 2,2,2trifluoroethanol (TFE, Acros America, 99.8%) were collected at 20 °C using an AVIV Stopped Flow 202SF CD Spectropolarimeter, using a total of five scans per sample, 1 nm bandwidth, and a 0.5 nm/s scan rate. The CD spectrometer was previously calibrated with d-10 camphorsulfonic acid. For all studies, protein samples in 100 µM TrisHCl, pH 7.5, in deionized distilled water were scanned from 185 to 260 nm. Initial CD studies examined a range of concentrations for each peptide (2, 4, 8, 12, 24 µM). For folding propensity studies, 12 µM peptide samples were titrated with either 2,2,2-trifluoroethanol (TFE, 99.8%, Acros America) at varying volume percentages.28,32 For metal ion binding studies, µL additions of CaCl2 stock solution (99.9% pure, Sigma-Aldrich) were added to the diluted stock solution to create a range of Asprich “3”/metal ion stoichiometries (2:1, 1:1, 1:4, 1:10, 1:20 protein/metal ion ratios). Spectra were obtained with appropriate background buffer subtraction performed (where applicable TFE, TrisHCl, CaCl2). The averaged spectra were smoothened using the SavitzkyGolay algorithm. Ellipticity is reported as mean residue ellipticity (deg cm2 dmol-1). Secondary structure estimation of Asprich “3” was performed using a constrained least-squares fit of the CD spectra with a 5-component reference spectra (R-helix, β-sheet, β-turn type I and II, random coil).33 The resulting fit was performed using IGOR Pro 6.0 and is reported as the fractional weight plus or minus the standard deviation of the five reference components.33

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Figure 2. Representative scanning electron micrographs of calcium carbonate crystals captured on silicon wafers. (A) 10 µM Asprich “3” sample. Here, heterogeneous rounded deposit sizes are noted, as well as the presence of rounded rhombohedral crystals on the surface of large mineral deposits or “fusions”. (B) 50 µM Asprich “3” sample. Note smooth surfaces of mineral deposits and the presence of deposit clustering. (C) 100 µM Asprich “3” sample. Note high density, small, irregular deposits and a large, amorphous-appearing deposit. Crystals obtained from control assays are shown in inset image (inset scale bar ) 50 µm).

Bioinformatics. Prediction of secondary structure units and identification of unfolded regions within the Asprich “3” primary sequence were conducted using the GLOBPLOT2.3,34 PSI-PRED,35 and SAMT9936 algorithms.

Results Modulation of Crystal Growth by Asprich “3”. Recent studies conducted with recombinant Asprich “g” reveal that this protein stabilizes ACC in solution and on the surfaces of nucleating calcite crystals.16 We would anticipate that similar functionalities exist with Asprich “3”. Thus, to establish the corresponding mineralization activity of Asprich “3”, we examined the effect of this protein on the nucleation of calcium carbonates (Figure 2), using protein concentrations that follow from our Asprich “a”-“g” terminal sequence studies.19 Under control conditions with no protein added, only calcite rhombohedral crystals were observed (Figure 2C, inset image). Similarly, in the presence of BSA, a nonspecific protein,31 only calcite crystals were observed (Figure S1, Supporting Information). However, in the presence of Asprich “3”, we observe that rounded calcium carbonates deposit on Si wafers (Figure 2). At 10 µM protein concentrations, these deposits are heterogeneous in size and in some instances we note the presence of large mineral aggregates or “fusions” that have roughened surfaces (Figure 2A). Upon closer examination it appears that the roughened surfaces of these deposits are formed by the presence of rounded rhombohedral crystals that are emerging from these “fusions”. At 50 µM protein concentrations the rounded deposits appear more homogeneous in dimension, with evidence of spherical cluster formation but no evidence of either rhombohedral crystal formation or the large “fusion” phenomenon (Figure 2B). In the SEM images, these deposits appear distorted from the spherical form, most likely as a result of settling out and adapting to or “flattening” out on the Si wafer surface. Note that amorphous minerals are expected to be isotropic, highly adaptable, and responsive in shape to their environment,24,26,37 which is consistent with our findings (Figure 2). Finally, at 100 µM Asprich 3 concentrations, mineral formation is severely limited, and we note the presence of irregular appearing granular deposits (Figure 2C), along with the occasional spherical deposit, which features irregular surfaces. Hence, the appearance of rhombohedral calcite crystals are progressively limited as a function of Asprich “3” concentrations. We note that calcite inhibition and round calcium carbonate deposits predominate in the presence of the recombinant Asprich “g” protein as well,16 and thus, the Asprich “3” protein qualitatively exhibits in vitro mineralization behavior similar to that of the “g” protein.

Figure 3. Representative Raman microscopy of mineralization assay material collected from control and Asprich “3” mineralization assays. The top row of light microscopic images represent the deposits that were examined. Scale bars ) 10 µm. The spectra peaks labeled as “C”, “V”, or “A” refer to calcite, vaterite, and aragonite, respectively. Note that the Si wafer peak at 519 cm-1 does not overlap with any of the calcium carbonate-specific peaks. The “x” peak represents absorbed impurities on the Si wafer. The Raman modes for synthetic aragonite, calcite, and vaterite can be found in Table 1.

Detection of Calcite, Vaterite, Aragonite, and ACC in Asprich 3 Mineral Deposits. Bulk X-ray diffraction of control and Asprich “3” assay mineral deposits confirm that various calcium carbonate polymorphs are present in Asprich “3” mineral deposits, with calcite being the predominant crystalline phase (Figure S2, Supporting Information). However, an ACC phase would not contribute to the bulk X-ray diffraction signal and thus cannot be detected using this method.24,26,38-40 With this in mind, we utilized microRaman spectroscopy to characterize the spherical deposits of the 10 and 100 µM Asprich “3” alongside the rhombohedral crystals of the control assay (Figure 3), with reference to Raman spectra obtained for calcite, aragonite, and vaterite (Table 1).19,38,39 In all of the Raman spectra, we note a strong peak at 519 cm-1 that arises from the

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Table 1. Raman Band Assignments for CaCO3 Polymorphsa mode -1

lattice mode (cm ) ν1, symmetric stretch (cm-1) ν2, out-of-plane bending (cm-1) ν3, asymmetric stretch (cm-1) ν4, in-plane bending (cm-1) overtones (cm-1)

calcite

vaterite

aragonite

156, 283 1086

118, 268, 301 1074, 1089

154, 208, 273 1086

874

854

1435

1445, 1485

1462, 1574

713

738, 750

704, 717

1749

1749

a

Taken from refs 19, 38, and 39. Reported values are for synthetic calcium carbonates.

Si wafer itself and minor peaks at 940-986 cm-1 that represent organic contaminants, such as volatile alcohols and amines adsorbed from the atmosphere and from trace compounds in our reagents.41,42 Surprisingly, we do not observe any Raman peaks that are protein-associated (i.e., peptide backbone amide groups modes at wavenumbers >1000 cm-1). Thus, the microRaman is not detecting an adsorbed or occluded Asprich “3” species associated with the rounded mineral deposits. We believe that this may be due to a number of factors: very low concentrations of adsorbed or occluded Asprich “3” species, microRaman sensitivity issues, or the possibility that these crystals form without the permanent adsorption of Asprich “3”. As expected, the control crystal exhibits the typical rhombohedral shape of calcite and the micro-Raman spectrum for this sample confirms this assignment (i.e., peaks at 152, 279, and 710 cm-1, Figure 3; Table 1).19,38,39 The symmetric stretch for calcium carbonates appears as a single peak at 1086 cm-1, which is consistent for calcite but not for vaterite or aragonite (Table 1). For the rounded 10 and 100 µM Asprich “3” mineral deposits we note the following changes in the Raman spectra. First, the broadening and intensity loss of the lattice mode 152, 279, and 710 cm-1 peaks indicates that well-ordered crystalline calcium carbonates such as calcite do not predominate in the rounded Asprich “3” mineral deposits (Figure 3).19,24,38,39 Simultaneously, the broadening of the carbonate symmetric stretching peak at 1086 cm-1 in the 10 and 100 µM Asprich “3” sample spectra is associated with ACC (Figure 3).24,40 Based upon the bulk X-ray diffraction, micro Raman spectra, and the SEM images (Figures 2, 3; Figure S2, Supporting Information), we conclude that Asprich “3” nucleates ACC deposits and calcite in vitro. The in vitro formation of ACC and calcite was also confirmed for recombinant Asprich “g”.16 Interestingly, we also observe a peak at 299 cm-1 that falls between the ranges for the lattice modes of vaterite (301 cm-1) and aragonite (208 cm-1), and this is consistent with the X-ray diffraction data for these samples (Figure S2, Supporting Information). This suggests two possibilities: either these two crystalline polymorphs are also forming in the presence of Asprich “3” or the ACC phase possesses lattice features that resemble vaterite and aragonite. Conformational Properties of Asprich “3”. The presence of structural disorder is essential for the function of IDPs,20-22 including those associated with biominerals.18,27,28,32,43 From our previous Asprich bioinformatics studies,18 we know that Asprich “3” hypothetically exists as an intrinsically disordered species (59%), with regions of disorder located at residues 6-20 and 39-59, as determined by the GLOBPLOT 2.3 prediction algorithm34 (see underlined regions in Figure 1). Using secondary structure prediction algorithms such as PSIPRED35 and SAM-T99,36 we confirm that sequence positions 6-20 and

Figure 4. CD spectra of 12 µM Asprich “3” in the presence of varying percentages of TFE (0-90%). Secondary structure classifications for 0% v/v and 75% v/v TFE were determined from CD spectra data set33 and are given in Table 2. Inset figure represents CaCl2 titration of 12 µM Asprich 3 at pH 7.5, 100 µM Tris-HCl. The Ca(II) CD spectra are presented as dashed lines and representative colors to demonstrate how the apo and Ca(II) CD curves overlap with one other.

39-59 do not correspond to folded domains (Figure 1). However, these predictive methods suggest that a helical domain may exist within the 14 AA helix-forming Glu, Ala, Val, Leu rich segment, -DDVEADAADLEEDV- of Asprich “3” (Figure 1). To experimentally verify the intrinsic disorder and folding propensity of Asprich “3”, we performed CD experiments in aqueous media and in the presence of the structure-stabilizing solvent, 2,2,2-trifluoroethanol (TFE). TFE has been used as a probe to confirm extrinsic stabilization capabilities of unfolded sequences via displacement of destabilizing water molecules at the peptide backbone.28,32 As shown in Figure 4, the CD spectra for Asprich “3” at 0% v/v TFE at neutral pH exhibits characteristics that are typical of an IDP. Specifically, there is a major (-) ellipticity band corresponding to the π-π* transition at 198 nm that is characteristic for random coil (RC) conformation.19,21,22,33 This is not unexpected, given the polyelectrolyte nature of Asprich “3” (Figure 1) and our current understanding of how polyelectrolyte domains destabilize protein structure.18,19,32,41 Comparison of this data with CD reference spectra33 reveals that Asprich “3” is predominantly RC (66%) and contains approximately 20-30% β-strand content, a small percentage of β-turn, and little if any R-helical content (Table 2). As TFE is introduced to the Asprich “3” sample, the π-π* ellipticity band broadens, becomes more positive in intensity, and experiences a red shift to 200 nm (Figure 4). This wavelength shift reflects a change in the conformational equilibrium from random coil toward other secondary structures.19,21,22,33 Based upon CD reference analysis33 for the 75% v/v TFE sample, this conformational equilibrium shift appears to favor β-turn structures (collectively 57%) at the expense of the β-strand and random coil structures (Table 2). Hence, the introduction of TFE leads to Asprich “3” structural reordering and an increase in nonrandom coil content. Interestingly, the unfolded RC state of Asprich “3” is unperturbed by the presence of Ca(II) ions (Figure 4, inset) and we intend to determine the relative Ca(II) binding affinity of this protein at a later date. Collectively, these results demonstrate that the Asprich “3” protein is an IDP and does not appreciably fold in the presence of Ca(II) ions. However, extrinsic stabilization of Asprich “3” is possible under the appropriate conditions (e.g., TFE) and leads to the formation of β-turn structures. Based upon our previous

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Table 2. Secondary Structure Estimates for Asprich “3”a R-helix

β-strand

β-turn type I

β-turn type II

random coil

0% TFE

75%TFE

0%TFE

75%TFE

0%TFE

75%TFE

0%TFE

75%TFE

0%TFE

75%TFE

0.0 ( 0.0

0.0 ( 0.0

0.27 ( 0.10

0.0 ( 0.0

0.06 ( 0.01

0.14 ( 0.05

0.0 ( 0.0

0.43 ( 0.15

0.66 ( 0.10

0.42 ( 0.15

a Percentages represent the fractional weight of each secondary structure type, determined by CD spectra database comparisons, as described in ref 33. β-Structures include β-sheet and β-turn types I and II.

structural studies of the highly conserved polyelectrolyte domains of Asprich “a”-“g”,18 we believe that regions such as the Pro, Lys, Arg -KPVFKRSLSDP-, and the Asp, Glucontaining polyelectrolyte sequences (Figure 1) act as destabilizers18,19,32,43 of Asprich “3” polypeptide structure at neutral pH and promote the unfolded conformation of this 61 AA protein. Polyelectrolyte destabilization effects may explain why the predicted helical domain, -DDVEADAADLEEDV- (Figure 1),34-36 does not adopt a helical conformation within Asprich “3” under the conditions utilized in our study (Figure 4, Table 2).

Conclusions This report details the preliminary characterization of Asprich “3”, the smallest member of the mollusk shell prismatic layer Asprich protein family. Like the recombinant Asprich “g” protein,15,16 Asprich “3” possesses the ability to inhibit or limit calcite formation in a concentration-dependent fashion and favors the formation of round calcium carbonate deposits (Figure 2). Further analysis of the Asprich “3” deposits confirm the presence of ACC along with calcite (Figure 3, Table 1; Supporting Information, Figure S2). ACC formation or rounded calcite deposits did not occur in the presence of BSA controls (Figure 2; Figure S1, Supporting Information), suggesting that Asprich “3” is a unique biomineralization protein that modulates both amorphous and crystalline calcium carbonate nucleation. The presence of vaterite and aragonite lattice features in the Asprich “3” mineral deposits (Figure 3; Figure S2, Supporting Information) is intriguing and suggests that either these polymorphs are forming in Asprich “3” assays or the ACC phase possesses some degree of vaterite- or aragonite-like lattice ordering. We intend to investigate this phenomenon in more detail at a later date. The Asprich “3” protein is intrinsically disordered (Figures 1 and 4; Table 2), predominantly RC (66%), and contains approximately 20-30% β-strand content with smaller percentages of β-turn and R-helix. We believe that the source of this conformational disorder is most likely the Pro, Arg, Lys cationic and the anionic Asp, Glu-containing regions (Figure 1), because polyelectrolyte sequences are known to be intrinsically disordered and induce conformational destabilization in the neighboring sequences which flank these regions.18,19 It is clear that Ca(II) ions alone cannot structurally stabilize this protein in solution (Figure 4), yet some degree of internal stabilization (transformation of RC and β-strand to β-turn, Table 2) does occur when TFE is introduced. Thus, the IDP Asprich “3” contains regions that can undergo conformational transformation, and these regions may stabilize or fold when confronted by a target other than Ca(II) ions that substantially minimizes the charge repulsion in these anionic regions. Conceivably, a mineral surface, ionic clusters, or other prismatic matrix proteins may be the preferred target(s) of Asprich “3”. At this time we do not know which sequence regions are conformationally transformable, and this feature will be determined using NMR spectroscopy.

There are a number of unanswered questions raised by our study. MicroRaman analysis could not detect adsorbed or occluded Asprich “3” protein associated with either ACC or calcite (Figure 3). This may be due to either very low concentrations of adsorbed or occluded Asprich “3” species, microRaman sensitivity issues, or the possibility that these crystals form without the need for Asprich “3” to remain adsorbed to the mineral phase. Thus, we do not know the mineral location of Asprich “3” and additional experiments will be required to determine if this protein resides on the surface and/or within these mineral deposits. Furthermore, we do not know the mechanism by which Asprich “3” induces ACC in vitro, nor do we know if the true in vivo function of Asprich “3” is to nucleate ACC during prismatic shell layer development. Similarly, the kinetics of Asprich “3”-mediated calcite crystal growth are unknown at present. These issues will be addressed in subsequent experiments. Acknowledgment. We thank Dr. Chunhua (Tony) Hu at the NYU Department of Chemistry for his help with X-ray data collection. This work was supported by funding from the National Science Foundation (DMR-0704148 to J.S.E., CHE0840277 to NYU), the J.D. Watson Investigator Program (NYSTAR Contract No. C050017 to L.E.), and Cornell’s Center for Materials Research (CCMR), the National Science Foundation Materials Research Science and Engineering Centers (MRSEC) program (DMR 0520404). Particular acknowledgment is made of the use of the Surface Analysis and Characterization facility of the CCMR. This paper represents contribution number 56 from the Laboratory for Chemical Physics, New York University. Supporting Information Available. Representative TEM images and electron diffraction pattern of calcium carbonate crystals formed in the presence of bovine serum albumin (BSA) collected by supernatant siphoning (Figure S1) and powder micro-X-ray diffraction spectra of control and Asprich 3 induced mineral deposits captured on Si wafers (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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