Polymorph Crystal Selection by n16, an Intrinsically Disordered Nacre

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Polymorph Crystal Selection by n16, an Intrinsically Disordered Nacre Framework Protein Christopher B. Ponce and John Spencer Evans* Laboratory for Chemical Physics, New York University, 345 East 24th Street, New York, New York 10010, United States

bS Supporting Information ABSTRACT: Framework proteins are a subclass of mollusk shell nacreassociated polypeptides that form supramolecular assemblies with β-chitin and other matrix proteins. These macromolecular assemblies manage the energetics of aragonite polymorph nucleation, and thus, there is keen interest in understanding the molecular characteristics of framework proteins. Here, we report the mineralization activity, oligomerization, and structural features of a recombinant framework nacre protein n16, isoform 3 (r-n16.3, Japanese pearl oyster Pinctada fucata). We find that r-n16.3 assembles in mineralization solutions to form spheroidal-fibril and mineralized thin film assemblies, in addition to spherical vaterite mineral deposits and aragonite single crystal deposits that possess unusual texture and layered morphologies. The oligomerization of r-n16.3 is spontaneous over the pH range 5
8.5, and protein particle sizes are observed to increase in radii when Ca(II) is present. Bioinformatics studies reveal that the r-n16.3 molecule is intrinsically disordered (random coil) and possesses residual α helix and β sheet structure. Experimentally, we confirmed that the secondary structure of apo-r-n16.3 assemblies is largely disordered (50% random coil, 20% β strand, 8% α helix). However, in the presence of high Ca(II) concentrations, we observe IDP disorder-to-order transformations that increase β turn structure and decrease random coil, α helix, and β strand contents. We conclude that r-n16.3 is an intrinsically disordered oligomeric nacre framework protein that nucleates vaterite and single crystal aragonite in vitro and possesses target-specific IDP disorder-to-order transformation capabilities in response to Ca(II).

’ INTRODUCTION In some mollusks, such as the Japanese pearl oyster, Pinctada fucata, the mineralized shell is comprised of two opposing layers of calcium carbonate known as the nacre (aragonite) and prismatic (calcite) layers.1
5 The material properties of each shell layer are different (nacre = fracture resistant; prismatic = puncture-resistant), and these properties arise in part from the presence of biomacromolecular assemblies in each shell layer.3
5 With regard to the nacre layer, the formation of aragonite instead of calcite is an intriguing process that has yet to be fully understood. However, it is clear that the nacre layer is assigned specific macromolecular components to achieve this feat. In particular, the three major biomacromolecular components that are believed to be responsible for the formation of the aragonite mineral phase are β-chitin polysaccharide,1,2,4,6 a silk-like fibroin protein that exists as a hydrogel,1,2,4 and an assemblage of intracrystalline and framework proteins.1,2,4
14 In vitro experiments have demonstrated that this multicomponent biomacromolecular system nucleates aragonite crystals.5,6,11,13 Thus, if we can deduce nacre component assembly and subsequent nucleation processes, then we will possess the necessary information that will enable the development of biomimetic approaches for manipulating crystal growth, designing supramolecular assemblies, and constructing biocomposites in the laboratory. r 2011 American Chemical Society

Given the complexity of the nacre system, it is instructional to first examine separate components and then advance to the study of multicomponent systems. With this approach in mind, we turn our attention to the framework protein family, a series of nacre proteins which bind to β-chitin polysaccharide to form a biomacromolecular complex that nucleates aragonite on the surface of this complex.3,5,6,13 These proteins are in contrast to the intracrystalline series of nacre proteins which nucleate aragonite within protein supramolecular assemblies.7,12,15 In P. fucata, three major framework nacre proteins have been identified. Two of these proteins, Pif97 and Pif80, represent cleavage products from a precursor Pif protein.5 A third framework protein, n16, is found in association with the Pif proteins and nucleates aragonite in the presence of Mg(II) ions in vitro.3 Unlike Pif, the n16 family contains no known post-translational modifications3 and consists of three isoforms (1,2,3) that exhibit slight differences in their N-terminal sequences.3,5 It is believed that n16 and Pif80/Pif97 assemble with β-chitin to form lamellar framework nucleation sheets or films within the nacre layer that induce aragonite formation and control crystal orientation.5 Part of this hypothesis has been confirmed using a 30 AA N-terminal Received: August 5, 2011 Revised: August 26, 2011 Published: August 29, 2011 4690

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Figure 1. Primary sequence of the r-n16.3 protein and GLOBPLOT2.3 prediction of intrinsically disordered and secondary structure regions. Secondary structure predictions are presented above the sequence (red arrow = β strand; blue rectangle = α helix; black line = random coil). Sequence regions outlined in black denote predicted regions of intrinsic disorder. Anionic (red) and cationic (blue) amino acids are indicated. The location of the n16N sequence is underlined in red (positions 1
30).10,17

sequence of the n16.3 isoform (n16N, Figure 1),6,10,16
19 which nucleates aragonite, vaterite, or ACC on β-chitin surfaces,6,16 or self-assembles to form lamellar polypeptide
aragonite complexes18 or spheroidal
fibril complexes that nucleate vaterite.19 Unfortunately, the experimental details of how the actual n16 protein participates in aragonite nucleation or supramolecular complex formation are currently lacking. To obtain more information regarding framework protein function, we have moved beyond peptidomimetics6,10,16
19 and created a recombinant version (bacterial expression) of the n16 isoform 3 protein (r-n16.3, Figure 1) and subsequently performed preliminary characterization of the in vitro mineralization activity, secondary structure, and self-assembly capabilities of this protein in the nonreduced state. We find that r-n16.3 preferentially nucleates vaterite and in limited instances generates layered or textured single crystal deposits of aragonite without the need for additives. Within these same assays, we note the presence of spheroidal
fibril deposits and protein films similar to those produced by the n16N peptide,17 and biophysical experiments confirm that r-n16.3 is an intrinsically disordered protein (IDP)20
22 that self-assembles above pH 5 to form nanometer-sized complexes in solution that increase in size and/or number when Ca(II) is present.

’ EXPERIMENTAL SECTION r-n16.3 Purification and Sample Preparation. The gene synthesis, cloning, bacterial expression, and purification of r-n16.3 were performed by GenScript USA (Piscataway, NJ, USA; http://www. genscript.com/) using their proprietary OptimumGene system and E. coli bacterial recombinant expression systems. The synthetic DNA was created from the complete n16.3 isoform sequence (Figure 1) (accession number Q9TW98)3 starting at residue 24 through 131 (108 AA). This oligo DNA omits the 23 AA membrane signal sequence. To this DNA sequence was incorporated a hybrid poly-(His)6 tag
enterokinase (EK) protease cleavage site at the N-terminus of the r-n16.3 sequence to facilitate the purification of r-n16.3 in a one-step fashion using high affinity Ni chelation chromatography. Following Ni elution, poly-(His)6 tag removal was accomplished via EK digestion and subsequent affinity capture of the EK enzyme. The final yield of purified r-n16.3 was approximately 0.8 mg/L LB culture from inclusion bodies, and the final protein purity was determined to be 94
96% (4%
20% gradient SDS-PAGE/Coomassie Blue staining, reducing conditions), with an apparent MW of 13 kDa (Figure S1, Supporting Information), which is close to the hypothetical MW of 12947 Da. The calculated pI of

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r-n16.3 is 4.56. A minor impurity (4
6%) was noted at 39 kDa and is suspected to be an oligomeric form (trimer) of r-n16.3. This minor impurity can be subsequently removed from the protein sample via ultrafiltration (Amicon Ultra 0.5, 30 kDa MWCO, Millipore Corporation, Billerica, MA, USA) at 14,000 rpm, 15 min, 25 °C, with collection of flow-through (r-n16.3) and discarding retained material (oligomer). Purified protein stock aliquots were stored in 50 mM Tris-HCl/10% v/v glycerol (pH 8.0) at
80 °C until needed. For subsequent experimentation, r-n16.3 samples (1
5 mg/mL as determined by Bradford protein assay using bovine serum albumin as a standard) were created by exchanging and concentrating appropriate volumes of stock solution into unbuffered deionized distilled water (UDDW) or other appropriate buffers using Amicon Ultra 0.5, 3 kDa MWCO. r-n16.3 Mineralization Studies. Using the same assay conditions employed in nacre protein
aragonite formation studies,6,12,15
18 we monitored the effect of r-n16.3 on calcium carbonate crystal growth. These mineralization assays are single stage and use solid (NH4)2CO3 that vaporizes over time and yields carbon dioxide gas which subsequently dissolves in the assay solution. Excess dissolution of CO2 increases the pH of the solution while producing CO32
ions.6,12,15
18 The initial pH of the assay solutions containing r-n16.3 peptide and 12.5 mM CaCl2 was found to be 4.0 and reached a value of 8.0
8.3 at the conclusion of the assay.6,12,15
18 These assay solutions contained either no protein (negative control) or final assay concentrations of 5, 10, and 25 μg/mL r-n16.3 protein. The collection procedure for SEM analysis of assay precipitates involved the use of Si wafer fragments (1 cm2 or less in size, “P” type [1 0 0], 20 Ohm-cm, 250
350 μm, Silicon Quest Intl., Santa Clara, CA) that were placed shiny side up at the bottom of assay wells prior to the start of the assay.17 For TEM analysis, we substituted Formvar-coated Au TEM grids for the Si wafers.17 Si wafers and Au grids containing assay deposits were washed with 50% ethanol/50% UDDW and then dried at 37 °C overnight. For SEM, dried Si wafer samples were coated with a thin layer of gold and then imaged using Hitachi S-3500N scanning electron microscopes at an accelerating voltage of 20 kV utilizing the Robinson backscatter image detector. Energy dispersive spectra (EDS) were collected on thin Au-coated samples using the Hitachi SEM equipped with PGT-Bruker X-ray microanalysis system at 20 kV. Transmission electron microscopy and electron diffraction measurements were also performed on Formvar-coated Au grids using a Philips CM12 transmission electron microscope at 120 kV. Electron diffraction spot patterns were indexed to calcium carbonates (aragonite) using the WWW-MINCRYST database (aragonite, card 5412).23,24 X-ray Diffraction. Washed and dried control and r-n16.3 Si wafer supports from mineralization experiments were analyzed using a Bruker D8 DISCOVER GADDS microdiffractometer equipped with a VANTEC-2000 area detector in a j 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 Kα = 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. Calcite, aragonite, and vaterite-specific X-ray diffraction data were obtained from the powder diffraction file (PDF), a database of X-ray powder diffraction patterns maintained by the International Center for Diffraction Data (ICDD), http://www.icdd.com/.

Dynamic Light Scattering Studies of r-n16.3 Oligomerization. The oligomerization of r-n16.3 was determined by measuring the hydrodynamic radius under different pH conditions (pH 5.0
8.5) and in the presence of Ca(II) using a Protein Solutions DynaPro MS/X dynamic light scattering (DLS) instrument. Sodium acetate and TrisHCl buffers (all 10 mM) were used to create the pH ranges 5
6 and 4691

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standard deviation of the five reference components. The β sheet, β turn type I, and type II are combined under the label “β structures”. The r-n16.3 sequence was also analyzed for the presence of disordered regions using the disorder prediction algorithm, GLOBPLOT2.3,29 which utilizes propensities to identify whether or not a given amino acid in a sequence is either random coil (RC) or a defined secondary structure.

’ RESULTS

Figure 2. Representative powder micro-X-ray diffraction spectra of Si wafers taken from protein-free (control) and r-n16.3-containing (10, 20 μg/mL) mineralization assays. Calcite (c), aragonite (a), and vaterite (v) reference peaks are denoted and were obtained from known data sets.23,24. 7
8.5, respectively.17 Stock solutions of CaCl2 (99.9%, Sigma/Aldrich) were utilized to create final Ca(II) concentrations of 12.5 mM for each protein sample at each investigated pH point. This Ca(II) concentration corresponds to that utilized in our mineralization assays.6,12,15
18 Prior to DLS measurements, all buffer/protein samples were prefiltered using 0.22 μm polyvinylidene fluoride syringe filters (Fisher Scientific) and then placed into quartz curvettes. Experimental conditions that were tested include protein concentration variations (5
50 μg/mL, pH 4.0/8.0, 25 °C), temperature dependence (5
45 °C, 25 μg/mL, pH 8.0), and pH variation (25 μg/mL, 25 °C, 4.0
8.5 for Ca(II); 4.0
11 for apo state) in the presence and absence of calcium. The samples were incubated at the indicated temperature for 10 min prior to measurements. Ten acquisitions were taken per trial. Analysis of the data and determination of the hydrodynamic radius (RH)25,26 were performed using the regularization analysis in the Dynamics v6.0 software provided with the instrument. By measuring the fluctuations in the laser light intensity scattered by the sample, the instrument is able to detect the speed (diffusion coefficient) at which the particles are moving through the medium. This value is converted to hydrodynamic radius (RH) using the Stokes
Einstein relation:25,26 D¼

kT 6πηRH

where D is the is the diffusion coefficient, k is the Boltzmann constant, T is the absolute temperature, η is the viscosity, and RH is the sphereequivalent hydrodynamic radius.26

Experimental and Predictive Structural Studies of r-n16.3. Circular dichroism (CD) spectrometry was utilized to determine the secondary structures present in r-n16.3 samples (25 μg/mL) at pH 8 in the presence and absence of Ca(II) in 10 mM Tris-HCl buffer, pH 8.0. We utilized Ca(II)/r-n16.3 mol stoichiometric ratios of 1:1, 10:1, and 6250:1 (which represents the 12.5 mM CaCl2 concentration utilized in our mineralization and DLS studies). For each sample, CD spectra were taken from the average of eight scans, with a scan rate of 0.5 nm/s from 185
260 nm, on an AVIV stopped flow 202SF CD spectropolarimeter.12,15,17,19 Spectra were obtained with appropriate background buffer subtraction performed, and averaged spectra were smoothed using the binomial algorithm with a smoothing setting of 50 and bounce end effects included in the IGOR Pro 6.0 program. Ellipticity is reported as mean residue ellipticity, θM (deg cm2 dmol
1). Secondary structure estimation of r-n16.3 was performed using a constrained least-squares fit of the spectra on a five-component model (α helix, β sheet, beta type I, type II, and random coil) utilized in the CD analysis software LINCOMB.27,28 The resulting fit was performed using IGOR Pro 6.0 and is reported as the fractional weight plus or minus the

Nucleation of Aragonite and Vaterite by r-n16.3. We investigated the effect of the nonreduced 108 AA r-n16.3 protein on mineral deposit formation captured on supports (Si wafers) which are placed at the bottom of assay wells prior to the start of these experiments. As shown in Figure 2, powder micro-X-ray diffraction analysis of captured assay deposits clearly shows that calcite and vaterite formation occurs in the presence of r-n16.3 (20 μg/mL and higher assay concentrations). Note that we found the formation of vaterite to be significantly lower in the control samples compared to the r-n16.3 assays. In addition to vaterite formation, the r-n16.3 powder diffraction spectrum also possesses three weak diffraction peaks which closely correspond to aragonite. These weak peak intensities indicate that the aragonite mineral phase is in low abundance relative to the vaterite in these assays. Hence, a mixture of calcite, aragonite, and vaterite form in r-n16.3 containing assays. These X-ray diffraction findings are verified by SEM and TEM analysis of the corresponding assay mineral deposits. In terms of morphology, we note that rhombohedral calcite crystals predominate in the control assays (Figure 3A), but spherical vaterite predominates in the r-n16.3 assays (Figure 3B). Interestingly, we did not observe any evidence of typical twinned crystal aragonite deposits6,16 in these assays. Rather, we noted the low occurrence of unusual mineral deposits (Figure 3C
E) that did not resemble either calcite or vaterite. These deposits are highly structured, are layered, and in some cases feature textured or patterned surfaces (Figure 3E), and we note that these deposits bear a qualitative resemblance to the layered lamellar aragonite structures formed in the presence of the n16N peptide.18 EDS confirm that these layered deposits are indeed mineralized (Figure 3F). However, due to the notoriously weak energy of the Cys thiol sulfur “S” peak and the possibility that r-n16.3 proteins are occluded within these unusual deposits, we could not confirm the presence of the r-n16.3 protein. TEM electron diffraction analysis revealed that these unusual deposits yield spot patterns that correlate with single crystal aragonite (Figure 4).30,31 Hence, the r-n16.3 protein influences the nucleation of calcium carbonate polymorphs, generating spherical vaterite and a minor component of morphologically unique aragonite. Evidence of r-n16.3 Spheroidal—Fibril and Film Formation in Mineralization Assays. In addition to the mineral deposits, we also observed the presence of spheroidal
fibril deposits which settled out onto Si wafers during the mineralization assay (Figure 5A). These qualitatively resemble the deposits formed by the n16N polypeptide under identical assay conditions17 and by other biomineralization proteins such as silicatein.32 As per our studies with n16N peptide,17 due to the small sizes of these deposits, we were unable to confirm via EDS whether or not these r-n16.3 deposits were mineralized. In addition to these spheroidal-fibril deposits, we also noted the presence of sheet-like or film deposits on regions of the Si wafers retrieved from r-n16.3 assays (Figure 5B). These films settle out 4692

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Figure 3. Scanning electron micrograph of mineral deposits captured by Si wafers in mineralization assays. (A) Backscatter image taken from proteinfree assay. Note predominance of calcite crystals. (B) Backscatter image taken from 20 μg/mL r-n16.3 assay. Here, vaterite crystals predominate. (C
E) Secondary electron images of representative layered or textured deposits captured by Si wafers in 20 μg/mL r-n16.3 assays. (F) Representative EDS spectra obtained for r-n16.3-generated layered/textured deposits, revealing high Ca content and thus confirming mineralization.

Figure 4. Representative transmission electron micrograph of layered/ textured mineral deposit (Figure 3D) and corresponding electron diffraction spot pattern. Indexing of this spot pattern correlates with aragonite [0 2 0], [1 1 0], and [2 0 0] Miller indices (WWWMINCRYST).24

during the assay and are observed to be thin and brittle, unlike the thicker, flexible films formed by n16N peptide.17 As shown in Figure 5B, these r-n16.3 films are often found adhering to mineral deposits. EDS measurements confirm that these sheet-like assemblies consist of r-n16.3 protein, as evidenced by the detection of protein-associated sulfur (S, Cys thiol) peaks (Figure 5C). But what distinguishes r-n16.3 films from n16N peptide films17 is that the r-n16.3 films actually mineralize (Figure 5C, note Ca levels), whereas films formed by the n16N peptide do not mineralize.17 The fact that r-n16.3 films are mineralized may explain why these films were found to be more brittle compared to the nonmineralizing n16N peptide films. We conclude that r-n16.3, like n16N, can oligomerize to form spheroidal
fibril deposits and protein films, with evidence that Ca(II) plays a role in the formation of r-n16.3 films. Mapping the Oligomerization of r-n16.3 in Solution. The detection of r-n16.3 spheroidal
fibril complexes and mineralized films led us to biophysical studies aimed at understanding the r-n16.3 assembly process. We note that the initial pH of the mineralization assay solutions containing r-n16.3 protein and 12.5 mM CaCl2 was found to be 5.0, and these solutions attain a pH value of 8.0
8.3 at the conclusion of the assay as the solution becomes saturated with carbonate vapor.20 Hence, either pH shift or Ca (II)
protein interactions may be responsible for triggering r-n16.3 assembly.

Figure 5. (A) Scanning electron micrograph of Si wafers recovered from 20 μg/mL r-n16.3 protein assays (secondary electron mode). Note the presence of spheroidal
fibril deposits on the wafer surface. (B) Scanning electron micrograph of filmlike deposits observed in 10 μg/mL r-n16.3 assays. Arrows indicate margins of protein film, and note the close correspondence between calcite crystals and the film. Note that drying procedures for SEM lead to cracking and porosity artifacts in the film. (C) Corresponding EDS spectra of r-n16.3 protein film, revealing the protein (sulfur, S) and Ca content of these films.

To clarify this, we used dynamic light scattering (DLS) methods to monitor the oligomerization of r-n16.3 as a function of pH (5
8.5) and in the presence and absence of Ca(II) (12.5 mM, corresponding to a mole Ca(II)/protein = 6250:1). We first examined the apo-assembly process as a function of pH. As shown in Figure 6A, the apoprotein spontaneously oligomerizes at pH g 5, forming particles with RH = 25
73 nm and polydispersity values >30%, which are indicative of heterogeneous particle size ranges.17,25,26 Note that similar results were obtained over the protein concentration range 5
50 μg/mL and the temperature range 5
45 °C (data not shown). The fact that the oligomer particle sizes are relatively constant over the pH range 5.5
8.5 indicates that pH variation does not dramatically affect protein particle size and reveals that r-n16.3 does not exist in a monomeric state within this pH range. Next, we examined the oligomerization process in the presence of 12.5 mM Ca(II). Here, the hydrodynamic radii of the r-n16.3 oligomers experienced a dramatic increase (RH = 91
127 nm; polydispersity values >15%) (Figure 6A), which indicates that Ca(II) promotes the formation of larger r-n16.3 particle sizes.25,26 These larger protein particles most likely form within our mineralization assays upon introduction of r-n16.3 into CaCl2 solutions prior to (NH4)2CO3 vapor introduction. 4693

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Table 1. Secondary Structure Estimates for r-n16.3 within Oligmeric Assemblies, pH 8.0a β turn

β turn

random

helix

β strand

type I

type II

coil

8 ( 1% 8 ( 1%

26 ( 7% 22 ( 6%

0 ( 4% 0 ( 4%

8 ( 10% 12 ( 9%

58 ( 11% 57 ( 10%

Ca(II) 10:1

7 ( 2%

29 ( 7%

0 ( 4%

12.5 mM Ca(II)

0 ( 1%

4 ( 6%

16 ( 3%

oligomer Apo-state Ca(II) 1:1

7 ( 11% 39 ( 8%

58 ( 11% 41 ( 8%

a

Percentages represent the fractional weight of each secondary structure type, determined by CD spectra database comparisons as described.27,28

Figure 6. (A) Dynamic light scattering analysis of r-n16.3 oligomerization (25 μg/mL) in various pH buffers in the presence and absence of 12.5 mM CaCl2, 25 °C. Note that similar results for apo-r-n16.3 were obtained over the concentration range 5
50 μg/mL and the temperature range 5
45 °C, indicating that the oligomerization of this protein is constant as a function of protein concentration and temperature (data not shown). (B) Circular dichroism spectra of 25 μg/mL r-n16.3, 25 °C, in 10 mM Tris-HCl, pH 8.0, in the presence and absence of CaCl2. The 12.5 mM Ca(II) concentration is identical to that utilized in the DLS and mineralization studies (Figures 2
4).

These DLS results are consistent with our EDS findings, namely, that r-n16.3 assemblies (i.e., films) are associated with significant Ca(II) content (Figure 5). We note that the primary sequence of r-n16.3 does feature carboxylate Asp and Glu residues (Figure 1), and we presume that these residues act as putative sites for Ca(II)-induced oligomerization. These findings are in contrast to the oligomerization behavior of the n16N peptide, which was found to be Ca(II)-independent.17 We believe that Ca(II) plays an important role in the formation of r-n16.3 oligomers and films. Secondary Structure of r-n16.3 within Oligmers. Predictive bioinformatics algorithms, such as GLOBPLOT2.3,28 can be used to identify regions of secondary structure and intrinsic disorder within protein sequences. This is particularly useful for self-associative proteins such as r-n16.3, which do not exist in a monomeric form under physiological conditions and thus cannot be readily assayed for secondary structure in a nonassociative state. Using this particular algorithm, we find that the r-n16.3 protein molecule is intrinsically disordered20
22,29 and possesses significant random coil segments (>50% of the total sequence) that are interrupted by short regions (10 AA or less) of secondary structure (α helix, β structures) (Figure 1). The most significant region of disorder is predicted to be at sequence positions 61
96, which encompasses the unusual Asn, Gly-rich repeat region.3 We note that earlier NMR studies of the monomeric n16N sequence revealed that this 30 AA peptide is intrinsically disordered and possesses residual β strand structure,19 and these features are correctly confirmed by the GLOBPLOT algorithm (Figure 1, underlined). Hence, we believe that the molecular

configuration presented in Figure 1 is a reasonable qualitative estimate of the secondary structure of r-n16.3 in the monomeric form. Using these bioinformatics calculations for comparison, we then experimentally determined the secondary structure of the r-n16.3 protein under oligomeric conditions using circular dichroism. We first examined r-n16.3 assemblies in the absence of Ca(II) at pH 8.0 (i.e., apo-oligomeric r-n16.3) (Figure 6A). Here, r-n16.3 possesses a strong (
) π
π* transition minimum band centered near 203 nm, corresponding to random coil structures in equilibria with other secondary structures.15,17,19,27,28 Estimation of secondary structure preferences using the reference data set27 reveals nearly 50% content of random coil, with approximately 20
30% β strand content and low percentages of α helix and β turn (Table 1).27 Interestingly, the experimentally determined random coil content is qualitatively similar to the values obtained via GLOBPLOT analysis (Figure 1).29 Hence, apo-r-n16.3 protein molecules within assembled oligomers exist as intrinsically disordered polypeptides that are primarily random coil in nature with residual β structure and α helical content. In other words, apo-oligomeric r-n16.3 is conformationally similar to the predicted structure of monomeric r-n16.3 (Figure 1). We conclude that the oligomerization of apo-r-n16.3 does not lead to significant structural change within participating protein molecules. Next, we investigated the secondary structure of r-n16.3 protein molecules within oligomeric assemblies in the presence of Ca(II), pH 8.0. Here, we were interested in learning if Ca(II)induced larger protein particles possessed similar or different secondary structure characteristics relative to the apo-assemblies and the predicted structure of monomeric r-n16.3. In the presence of low levels of Ca(II) (i.e., Ca/protein = 1:1, 10:1), we note little if any perturbation in the structural features of selfassociated r-n16.3 molecules relative to the apo-state (Figure 6B, Table 1), as evidenced by only minor changes in ellipticity intensities and no emergence of new ellipticity bands. Thus, low levels of Ca(II) have no observable effect on the secondary structure of protein molecules within oligomeric assemblies. However, in the presence of higher Ca(II) concentrations (i.e., 12.5 mM Ca(II), corresponding to a Ca(II)/protein = 6250:1 utilized in our DLS and mineralization studies), we note a significant decrease in ellipticity band intensities, which we attribute to the formation of larger r-n16.3 protein particles (and increased scattering phenomena) and potential salting-out effects that occur under these conditions (Figure 6A).27 The CD spectrum for this particular sample features weak (
) (195 nm, 208 nm, 222 nm) and (+) (198 nm, 215 nm) ellipticity bands, and the corresponding secondary structure analysis indicates a significant decrease in helical, β strand, and random coil content and a corresponding increase in β turn content relative to the 4694

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Crystal Growth & Design apo-state (Table 1).27,28 In other words, Ca(II)-oligomeric r-n16.3 is conformationally different from both the predicted structure of monomeric r-n16.3 (Figure 1) and the experimentally determined structure of protein molecules within apooligomeric r-n16.3 (Figure 6B, Table 1). Therefore, the high Ca(II) content found in our mineralization assays (Figures 2
5) not only promotes larger protein particle sizes (Figure 6A) but also leads to an IDP disorder-to-order transformation (i.e., random coil
β strand to β turn) within protein molecules that comprise r-n16.3 oligomers which form during the mineralization process (Figure 6B, Table 1).

’ DISCUSSION We have characterized a recombinant form of the n16 protein, isoform 3 (Figure 1). r-n16.3 possesses no post-translational modifications, and we note that no known post-translational modifications have been reported for n16.3,5 Using standard carbonate vapor mineralization assays,15
18 we observe that r-n16.3 possesses two traits that are associated with the n16N peptide sequence.17 First, both species promote the formation of vaterite over calcite (Figures 2 and 3).6,16,17 Second, both polypeptides infrequently generate aragonite deposits (Figures 3 and 4) that possess unusual morphologies featuring surface texture and evidence of layering.18 The n16N
aragonite layering phenomenon was attributed to the presence of layered polypeptide sheets which nucleated mineral layers,18 and we suspect that r-n16.3 aragonite deposits (Figures 3 and 4) may also contain protein layers or films, but we were unable to confirm this with our present methodology (Figure 3) and thus additional experimentation will be required to verify protein occlusion. Nonetheless, the functional similarities between r-n16.3 and n16N indicate that the 30 AA N-terminal region of r-n16.3 is partly responsible for the polymorph selection activity of r-n16.3. Given that n16.3 is a framework protein3,5 that combines with Pif proteins and β chitin polysaccharide to generate aragonite,5 we emphasize that our mineralization results solely reflect an in vitro scenario involving only r-n16.3 in solution, and additional studies will be required to ascertain how r-n16.3 controls mineralization when associated with β-chitin and/or Pif proteins. Nonetheless, it is clear that r-n163, like the native n16 protein3,5 and other reported nacre protein sequences,12,13,15 exerts control over polymorphism yet does not require additional additives (other proteins, Mg2+) to do so. r-n16.3 represents a model nacre framework protein that will be used to understand nacre component assembly and polymorph selection processes in more detail. In many nacre-associated protein systems, the process of aragonite formation occurs in tandem with the oligomerization of these proteins.6,12,15,17,18 Our current data reveals that r-n16.3 possesses self-assembly capabilities as well, forming spheroidal
fibril deposits and films during the mineralization assay period (Figure 5). However, at this time we have not established that r-n16.3 deposits or films have nucleation capabilities, and this must await further experimentation. We do note that the formation of spheroidal
fibril deposits and films is a trait that is also shared by the n16N peptide sequence,17 and thus, we believe that this 30 AA domain is a participant in r-n16.3 polymorph selection AND protein oligomerization. The formation of r-n16.3 protein particles and films arises from the spontaneous oligomerization of this protein in solution over the pH range 5
8.5 (Figure 6A). As per n16N,17 it is likely that electrostatic interactions (salt bridging) between anionic (Asp, Glu, deprotonated

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over the pH 5
8.5 range; Cys-SH, deprotonated at pH > 8) and cationic groups (His, Arg, Lys, protonated within the same pH range) (Figure 1) are responsible for r-n16.3 oligomerization. The effect of Ca(II) on r-n16.3 particle sizes (Figure 6A) and the presence of Ca(II) mineral within r-n16.3 protein films (Figure 5) suggests that Ca(II) plays a central role in the formation of r-n16.3 assemblies. This is in contrast to earlier observations obtained for the n16N peptide, where we observed that Ca(II) does not change n16N peptide particle sizes and that n16N peptide films do not mineralize.17 We believe that these discrepancies can be attributed to the additional proteomic information (Figure 1) that exists within the r-n16.3 protein but omitted from the N-terminal 30 AA n16N peptide. In other words, we postulate that downstream of the n16N domain there exist putative sites for Ca(II)-induced agglomeration and calcium carbonate nucleation (Figure 1). Potentially, one of these regions may be the 30 AA C-terminal domain (Figure 1), which was previously shown to modulate calcium carbonate nucleation in vitro.10 Further research will be required to determine if this C-terminus or other sequence regions within r-n16.3 participate in aragonite nucleation and protein self-assembly. Finally, we believe that the disordered, unfolded state of apo-rn16.3 (Figures 1 and6B) offers conformational freedom19 and structural adaptation20
22,33
36 that would allow interactions and multiple contacts to occur between an array of different matrix targets, such as other r-n16.3 molecules,3,18 other nacre matrix proteins (Pif),5 β chitin,3,5,6,16 or the mineral phase itself.6,16,18 Unfolded or disordered conformations also offer advantages in terms of entropy: the disorder-to-order transition that occurs upon unfolded IDP binding to targets would decrease conformational entropy and make highly specific interactions possible during nacre mineralization.20,21,33,36 Interestingly, apooligomeric r-n16.3 exhibits no evidence of folding or structural reorganization, indicating that r-n16.3 protein
protein interactions are insufficient to perturb internal protein structure. This is consistent with a key tenet of the intrinsic disorder hypothesis, which states that some IDP molecules retain their disordered state when bound to one series of targets (i.e., other r-n16.3 molecules) but undergo disorder-to-order transformations only when they encounter another series of targets.20
22 In contrast, high Ca(II) concentrations, such as those found in mineralization assays (Figures 1
3), may be one of the true “targets” for r-n16.3 oligomers (Figure 6B). Under these conditions, the oligomerization of r-n16.3 shifts to larger particle sizes (Figure 6A), and simultaneously, r-n16.3 protein molecules within these assemblies undergo disorder-to-order transitions, leading to a reduction in random coil, α helix, and β strand structure and an increase in β turn content (Figure 6B). This finding may have relevance to the mineralization of these films (Figure 5) and possibly the formation of aragonite (Figure 2). Future experiments will hopefully address this possibility.

’ ASSOCIATED CONTENT

bS

Supporting Information. 4
20% SDS/PAGE gradient gel of Ni-column purified r-n16.3 (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: 2129989605. Fax: 2129954087. E-mail: jse1@nyu. edu. 4695

dx.doi.org/10.1021/cg201015w |Cryst. Growth Des. 2011, 11, 4690–4696

Crystal Growth & Design

’ ACKNOWLEDGMENT We thank Drs. Eric Roth (TEM), John Ricci (SEM/EDS), and Chunhua “Tony” Hu (X-ray Diffraction) of New York University for their assistance in the experimental phases of this work. This research was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award DE-FG02-03ER46099, and represents contribution number 62 from the Laboratory for Chemical Physics, New York University. ’ ABBREVIATIONS n16N = 1-30 AA N-terminal domain of the Pinctada fucata Japanese pearl oyster nacre protein, n16.3; r-n16.3 = recombinant (E. coli) P. fucata n16 framework nacre protein, isoform 3, 108 AA; IDP = intrinsically disordered protein; UDDW = unbuffered deionized distilled water; DLS = dynamic light scattering; EDS = energy dispersive spectra; ACC = amorphous calcium carbonate; AP7 = aragonite protein 7, Haliotis rufescens; PFMG1 = Pinctada fucata mantle gene product 1; CD = circular dichroism spectrometry; PDF = powder diffraction file; EK = enterokinase ’ REFERENCES (1) Mann, S., Webb, J., Williams, R. J. P., Eds. In Biomineralization: Chemical and Biochemical Perspectives; VCH: Weinheim, Germany, 1989. (2) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford Press: New York, NY, 1989. (3) Samata, T.; Hayashi, N.; Kono, M.; Hasegawa, K.; Horita, C.; Akera, S. FEBS Lett. 1999, 462, 225–232. (4) Nudelman, F.; Gotliv, B. A.; Addadi, L.; Weiner, S. J. Struct. Biol. 2006, 153, 176–187. (5) Suzuki, M.; Saruwatari, K.; Kogure, T.; Yamamoto, Y.; Nishimura, T.; Kato, T.; Nagasawa, H. Science 2009, 325, 1388–1390. (6) Keene, E. C.; Evans, J. S.; Estroff, L. A. Cryst. Growth Des. 2010, 10, 1383–1389. (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. (8) Fu, G.; Qiu, S. R.; Orme, C. A.; Morse, D. E.; DeYoreo, J. J. Adv. Mater. 2007, 17, 2678–2683. (9) Shen, X.; Belcher, A. M.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. J. Biol. Chem. 1997, 272, 32472–32481. (10) Kim, I. W.; DiMasi, E.; Evans, J. S. Cryst. Growth Des. 2004, 4, 1113–1118. (11) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67–69. (12) Amos, F. F.; Evans, J. S. Biochemistry 2009, 48, 1332–1339. (13) Gries, K.; Heinemann, F.; Gummich, M.; Ziegler, A.; Rosenauer, A.; Fritz, M. Cryst. Growth Des. 2011, 11, 729–734. (14) Marie, B.; Marin, F.; Marie, A.; Bedouet, L.; Dubost, L.; Alcaraz, G.; Milet, C.; Luquet, G. ChemBioChem 2009, 10, 1495–1506. (15) Amos, F. F.; Destine, E.; Ponce, C. B.; Evans, J. S. Cryst. Growth Des. 2010, 10, 4211–4216. (16) Keene, E. C.; Evans, J. S.; Estroff, L. A. Cryst. Growth Des. 2010, 10, 5169–5175. (17) Amos, F. F.; Ponce, C. B.; Evans, J. S. Biomacromolecules 2011, 12, 1883–1889. (18) Metzler, R. A.; Evans, J. S.; Kilian, C. E.; Zhou, D.; Churchill, T. H.; Appathurai, P. N.; Coppersmith, S. N.; Gilbert, P. U. P. A. J. Am. Chem. Soc. 2010, 132, 6329–6334. (19) Collino, S.; Evans, J. S. Biomacromolecules 2008, 9, 1909–1918. (20) Uversky, V. N. Protein Sci. 2002, 11, 739–756.

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