Selective synergism created by interactive nacre framework

3 days ago - In the nacre layer of the Pinctada fucata oyster shell there exists a multi-member proteome, known as the framework family, which regulat...
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Selective synergism created by interactive nacre framework-associated proteins possessing EGF and vWA motifs: Implications for mollusk shell formation. Gaurav Jain, Martin Pendola, Yu-Chieh Huang, Denis Gebauer, Eleni Koutsoumpeli, Steven D. Johnson, and John Spencer Evans Biochemistry, Just Accepted Manuscript • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018

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Biochemistry

Selective synergism created by interactive nacre frameworkassociated proteins possessing EGF and vWA motifs: Implications for mollusk shell formation.

Gaurav Jain,1† Martin Pendola,1† Yu-Chieh Huang,2 Denis Gebauer,2 Eleni Koutsoumpeli,3 Steven Johnson,3 and John Spencer Evans1†*

1

Laboratory for Chemical Physics, Center for Skeletal and Craniofacial Biology, New York University, 345 E. 24th Street, NY, NY, 10010 USA. 2

Department of Chemistry, Physical Chemistry, Universität Konstanz, Universitätstrasse 10, Konstanz D-78457, Germany. 3

Department of Electronic Engineering, University of York, Heslington, York, YO105DD, United Kingdom

*To whom correspondence should be addressed: John Spencer Evans, Laboratory for Chemical Physics, Division of Basic Sciences and Center for Skeletal and Craniofacial Medicine, New York University College of Dentistry, 345 E. 24th Street, New York, NY, 10010. Tel.: (212) 998-9605; Fax: (212) 9954087. Email: [email protected].

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ABBREVIATIONS r-n16.3 = recombinant Pinctada fucata framework protein 16, isoform 3; r-Pif97 = recombinant Pinctada fucata framework protein 97; Pif80 = Pinctada fucata framework protein 80; QCM-D = quartz crystal microbalance with dissipation monitoring; PNC = prenucleation cluster; ACC = amorphous calcium carbonate; FIB = focused ion beam sectioning. FSC-A = forward scattered component; SSC-A = side scattered component; vWA = von Willebrand Factor Type A; EGF = epidermal growth factor

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ABSTRACT

In the nacre layer of the Pinctada fucata oyster shell there exists a multi-member proteome, known as the framework family, which regulates the formation of the aragonite mesoscale tablets and participates in the creation of an organic coating around each tablet. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights we have created a proportionally-defined combinatorial model consisting of two recombinant framework proteins, r-Pif97 (containing a von Willebrand Factor Type A domain) and r-n16.3 (containing an EGFlike domain) whose individual in vitro mineralization functionalities are distinct from one another. We find that at 1:1 molar ratios r-Pif97 and r-n16.3 exhibit little or no synergistic activity regarding modifying existing calcite crystals. However, during the early stages of nucleation in solution, we note synergistic effects on nucleation kinetics and ACC formation/stability (via dehydration) that are not observed for the individual proteins. This selective synergism is generated by Ca2+ - mediated protein – protein interactions (~ 4 molecules of r-n16.3 per 1 molecule of r-Pif97) which lead to the formation of nucleation-responsive hybrid hydrogel particles in solution. Interestingly, in the absence of Ca2+ there are no significant interactions occurring between the two proteins. This unique behavior of the framework-associated n16.3 and Pif97 proteins suggests that the Asp, Glu-containing regions of the vWA and EGF-like domains may play a role in both nacre matrix formation and mineralization.

Keywords:

Pearl oyster, nacre, biomineralization, n16.3, Pif97, amorphous calcium carbonate, pre-

nucleation clusters, QCM-D, flow cytometry, SEM, potentiometric titration, smart hydrogels

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INTRODUCTION The nacre layer in marine invertebrate shell is a composite material system that is optimized for fracture toughness.1-3 In most mollusks the nacre layer consists of a “brick-and-mortar” construction where mesocrystal aragonite tablets are arranged in parallel layers.1-3 Each tablet arises from the assembly of calcium carbonate nanoparticles during shell development.4-7 As a composite, the nacre is tougher than pure aragonite1-3 and its layered structure contributes to the luster and strength of the nacre.27

The nacre formation process requires the synthesis and assembly of a protein network.8-14 The resultant

material is highly ordered, integrated, and possesses mesoscale bulk properties (fracture resistance, color, lustrous appearance) that originate from nanoscale components and their singular properties.1-7 Although this nano-to-mesoscale aragonite assembly process is not well understood, studies have shown that protein families or proteomes play an important role in the nano-to-mesoscale assembly of the nacre.8-27 One of the regions in the nacre where this becomes evident is the framework organic coating that surrounds each mesocrystal tablet.8-17 This coating is gel-like, promotes nucleation, and consists of the polysaccharide, beta-chitin, and a proteome known as the framework or lamellar proteome.8-27 Although the role of the framework proteome is not well understood, previous studies have established that these proteins bind to beta chitin polysaccharide21,23,24 and can act as an elastomeric layer that protects the aragonite tablets from fracture.1-8 Furthermore, certain members of this proteome, namely, n16.38-13,17-22 and Pif97,8-12,14,23-27 (Figure 1) are co-expressed during shell development and participate in nucleation, with Pif97 acting as a calcite inhibitor23-27 and n16.3 acting as a calcite inhibitor, directional growth modulator, as well as an aragonite organizer and promoter. 17-22 Both proteins are intrinsically disordered, with Pif97 containing an interactive von Willebrand Factor Type A (vWA) motif25 and

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Biochemistry

Figure 1. Primary amino acid sequences of Pinctada fucata n16.3 (UniProtKB - Q9TW98 (MA163_PINFU) and Pif97 (cleavage product derived from the Pif177 gene, Swiss-Prot: C7G0B5.1; GenBank: BAH97338.1), with membrane leader sequences deleted. Anionic (red) and cationic (blue) sidechains are noted. The vWA domain of Pif97 is located at F23-Y161; the EGF-like alpha helical domain of n16.3 is located at D27-L60.

n16.3 containing EGF-like interactive helical motif (20-30% homology) found in knottin sequences.19 Both n16.3 and Pif97 are hydrogelators that form “smart” hydrogel protein particles that are responsive to solution conditions, and these hydrogels dramatically increase in dimension when Ca2+ is introduced.22,25 Given that Pif97 and n16.3 appear in the nacre matrix simultaneously,8,9,11,12 there is the possibility that both proteins work together and/or interact with one another during the development of the nacre layer. However, direct evidence of protein-protein interaction amongst these proteins has not yet been established, and thus our understanding of framework protein mediation of nacre mineralization is incomplete.

Recently, we introduced combinatorial recombinant protein studies where we investigated the effects of defined molar ratios (1:1) of nacre and pearl-associated proteins on in vitro ACC and calcite nucleation, and, verified protein-protein interactions within each pair.28,29 We found that the 1:1 molar

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ratio combination elicits the greatest response in mineral formation,28 and that the proteins exhibited noncovalent binding to one another, forming hybrid protein hydrogels that promoted synergistic effects on either the early and/or later stages of nucleation and crystal growth.28,29 Using this approach, we turned our attention to the two well-characterized members of the P. fucata framework proteome, n16.3 (MW = 12.9 kDa, 108 AA, pI = 4.82) 8-13,17-22 and Pif97 (97 kDa, 525 AA, pI 4.65) 8-12,14,23-27 (Figure 1) to determine if these framework sequences interact with one another and if functional synergism is exhibited during the mineralization process. Besides addressing protein-mediated biomineralization, n16.3 and Pif97 can serve as a model system for understanding several general biochemical phenomena, such as the role that vWA- and EGF motif-containing proteins play in forming protein complexes, mineral deposits, as well as the interactive capabilities of co-expressed matrix proteins. Using recombinant variants of both proteins (r-n16.3, r-Pif97),19,20,22,25 in vitro mineralization microassays, and biophysical techniques, we find that when both proteins are present together in a 1:1 molar ratio, they do not substantially deviate from their individual functionalities regarding modification of existing calcite crystals. However, during the early stages of nucleation, the 1:1 molar mixture synergistically increases the ACC nucleation time (kinetics) and alters the dehydration kinetics of nucleation intermediates. We also note that only when Ca2+ is present, both proteins become interactive, with an estimated 4 molecules of r-n16.3 interacting with every 1 molecule of r-Pif97, and we note that these interactions generate a hybrid hydrogel phase. Thus, the framework-associated n16.3 and Pif97 matrix proteins are selectively interactive and exhibit limited synergistic effects on the calcium carbonate mineralization process. This further demonstrates the involvement of vWA- and EGF-like sequences in protein-protein complexation and illustrates important traits for inhibiting calcite growth, thereby allowing aragonite to form in the environment of the framework layer.

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Biochemistry

EXPERIMENTAL PROCEDURES

Sample preparation. The expression, preparation and purification of recombinant n16.3 (r-n16.3, MW = 12947 Da)19 and Pif97 (r-Pif97, MW = 58808 Da)25 were performed as described previously. For subsequent experimentation, protein samples 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 concentration filters.19,25 In vitro micro-mineralization assays. Calcite-specific mineralization microassays were conducted by mixing equal volumes of 20 mM CaCl2*2H2O (pH 5.5) and 20 mM NaHCO3 / Na2CO3 buffer (pH 9.75 in Milli-Q Type I ultrapure water 0.22 µm filtered) to a final volume of 500 µL in sealed polypropylene tubes and incubating at room temperature for 1 hr.19,20,22,25 The final pH of the reaction mixture was measured and found to be approximately 8.0 - 8.2.19,20,22,25 Individual aliquots of r-n16.3 and r-Pif97 stock solutions were simultaneously added to the calcium solution prior to the beginning of the reaction, with final assay concentrations of each protein to be 5 μM (i.e., 10 μM total protein concentration). Mineral and protein deposits formed during all assays were captured on 5 x 5 mm Si wafer chips (Ted Pella, Inc.) that were placed at the bottoms of the vials. Upon completion of the mineralization assay period, the Si wafers were rinsed thoroughly with calcium carbonate saturated methanol and dried overnight at room temperature prior to analysis.19,20,22,25 Imaging of the Si wafers extracted from the mineralization assays was performed using a Merlin (Carl Zeiss) field emission SEM (FESEM) using either an Everhart-Thornley type secondary electron detector (SE2) at an accelerating voltage of 3 kV and a probe current of 100 pA. Prior to analysis, samples were coated with iridium (4nm layer) using a Cressington 208HR sputter coater with thickness controller.

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Focused ion beam sectioning of crystals. Using a Zeiss Auriga Small Dual-Beam FIB-SEM, imaging of internal crystal morphology was performed on crystals retrieved from 1:1 r-n16.3 : r-Pif97 simultaneous mixing assays. For these analyses all samples were first coated with 4 nm iridium prior to SEM imaging, then coated with 50 nm of Au prior to performing FIB.25,28,29 A 30 kV 120 pA gallium ion beam was oriented perpendicular to the sample by tilting the sample stage to 54˚ and utilized to mill 15 nm serial cross-sections. SEM images of cross-sectioned surfaces were then obtained using a 2.0 kV 600 pA electron beam and a secondary electron detector at a working distance of 5.0 mm. Images of surfaces containing electron beam damage were created for comparison to images of undamaged surfaces but were not used for the purposes of discussion in this publication. Images were taken shortly after cross sectioning to limit the exposure of the uncoated surfaces to the electron beam.25,28,29 Calcium potentiometric titrations. A computer-controlled titration instrument manufactured by Metrohm was utilized for the quantitative potentiometric titration experiments.25,28,29,33-37 The setup includes two Titrando devices (Titrando 809) controlling three dosing devices (800 Dosino) for dosing CaCl2, NaOH and HCl (10 mM), respectively. NaOH and HCl were used for balancing the ion binding between Ca2+ and carbonate species as well as out-diffusion of CO2, respectively.33-37 Calcium potential and pH values were monitored by utilizing one Ca(II) ion-selective electrode (Metrohm No. 6.0508.110) and one pH electrode (Metrohm No. 6.0256.100), respectively. Based on in-situ Ca(II) ion-selective potentiometric measurements at constant pH levels, the quantitative information can be assessed. During a titration experiment, CaCl2 (10 mM) was continually titrated into carbonate buffer (10 mM, 10 mL) at a constant rate of 20 μl /min and the pH was kept constant at pH 8.5 by automatic counter-titration of NaOH and HCl.33-37 In the presence of 50 nM and 500 nM r-Pif97, r-n16.3 as well as the 1:1 mixture, the potentiometric titrations were performed in 10 mL carbonate buffer at pH 8.5, respectively. The protein mixtures were simultaneously introduced to the apparatus and contained 50 nM and 500 nM of each

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Biochemistry

protein, thus the total protein concentrations were 100 nM and 1000 nM, respectively. Imaging and flow cytometry of individual and hybrid hydrogel particles. Using flow cytometry,38-42 the physical state of r-n16.3, r-Pif97, and 1:1 r-n16.3 : r-Pif97 hydrogel particles were studied in parallel under the following conditions: a) 10 mM HEPES, pH 8.0; b) 10 mM HEPES, 10 mM CaCl2, pH 8.0. Protein concentrations were 5 µM as per the micromineralization experiments. Samples were constituted and allowed to sit for 5 min prior to analysis. Particle measurements were performed using a multiparameter cell analyzer BD LSRFortessa (BD Biosciences, USA) with a sensitivity in the 1.5 – 2.0 µm range and resolution within the 5-20 µm range for both FSC and SSC parameters.38-42 Each sample solution (150 µL) was analyzed at a continuous flow rate of 25 µL/min using four laser excitation wavelengths of 405 nm, 488 nm, 561 nm, and 640 nm to register two light-scattering parameters (Forward Scattered Component or FSC-A and Side-Scattered Component or SSC-A; detailed definitions can be found in previous reports)38-42 and the number of events for each sample. Data was collected using the BD FACS DiVa software (BD Biosciences, USA) and processed using FlowJo software (TreeStar, OR, USA). In Figure 6 the reader will note that some of the data points lie as a continuous series at the chart limits. This is due to presence of real protein hydrogel particles that are smaller than the 5 µm threshold yet are detected and compensated via signal amplification by the instrument.38-42 This compensation leads to the continuous display of data points along the edges of a given chart due to the presence of non-resolvable small protein particles in the sample. QCM-D Interaction Studies. Quartz crystal microbalance with dissipation monitoring (QCM-D) experiments were performed using a Q-sense E4 system from Biolin Scientific. Gold-coated AT-cut quartz sensors were used (QSX 301, Biolin Scientific, Stockholm, Sweden), for which the fundamental frequency was 4.95 MHz ± 50 kHz.28,29 Initially, the sensors were placed successively in solutions of Hellmanex III (2%) and ultrapure Milli-Q water (twice) and sonicated in each bath for 10 min, with the

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active side facing upward in all instances. The sensors were then dried with N2 gas and replaced in the UV–ozone cleaner for 30 min. Finally, the sensors were left to soak in 100% ethanol for approximately 30 min and dried with N2 gas before installation in the flow modules (QSense E4, QFM 401, Biolin Scientific, Stockholm, Sweden). The QCM-D flow chambers were flushed with ultrapure Milli-Q water before each measurement, until a stable baseline was established ( r-n16.3 > r-Pif97, i.e., the 1:1 r-n16.3 : r-Pif97 mixture is synergistic in terms of inhibition; (3) When it comes to the stabilization of initially formed, more soluble ACC phases, we note that r-n16.3 and 1:1 r-n16.3 : r-Pif97 are nearly equivalent in their ability to initially promote the formation of more soluble ACC phases, particularly, at 500 nM. However, for the 1:1 mixture at 500 nM (Figure 5B, green curve), the post-nucleation development is much more curved and is indicative of a synergistic effect, whereas for r-n16.3 alone (Figure 5B, blue curve) it transitions into a linear regime quickly. (4) Given that the nucleation of solid amorphous phases proceeds via dehydration/solidification of dense mineral droplets formed directly from PNCs upon liquid/liquid demixing,37 and, that calcium carbonate-associated protein “smart” hydrogels bind and influence the movement and exchange of water39 and ions,38 the hydrogels created by the 1:1 framework nacre protein mixture are synergistic vis a vis the corresponding dehydration kinetics of nucleation intermediates.

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Ca2+ ions promote the formation of hybrid hydrogel particles. The selective effects that the 1:1 r-n16.3 : r-Pif97 sample exert on the early stages of the nucleation process (Figures 4,5, Table 1) suggest that the hydrogel particles in this mixture may have changed, such that they now function differently from the hydrogel particles formed by the individual proteins. A similar phenomenon was observed for pearl nacre-associated proteins r-PFMG1 and r-PFMG2.29 To determine if this is the case we utilized a lightscattering technique, flow cytometry,38-42 to probe the particle size distributions and granularities for the 1:1 r-n16.3 : r-Pif97 sample and compare these values to those obtained for the individual protein components. In flow cytometry we can monitor two light scattering parameters for particles under constant flow: 1) forward scattered light component (FSC, x-axis) to determine particle size distribution; and 2) side-scattered light component (SSC, y-axis) to measure changes in refracted and reflected light that results from variations in particle granularity or internal structure (further information can be found in the legend to Figure 6).38-42 As shown in Figure 6, individual r-n16.3 and r-Pif97 hydrogels each have unique particle size distributions yet similar particle granularities (i.e., internal structure) at pH 8.0 in the absence of Ca 2+ (apo-state). When these two proteins are present in a 1:1 mixture, we note that the particle granularities remain relatively constant but the particle size distributions shift to a value that approximates the average value for both individual proteins. This would suggest that the hydrogel particles generated by both proteins are co-existing but not combining in the absence of Ca2+.38-42 However, in the presence of 10 mM CaCl2,10 mM HEPES, pH 8.0, there are significant differences in both the particle size distributions and particle granularities for each individual protein, and in the 1:1 r-n16.3 : r-Pif97 sample, these individual hydrogel particle properties appear to merge together. From these results we conclude that the protein hydrogel particles that form in the presence of 1:1 r-n16.3 : r-Pif97 in Ca2+ buffered media are hybrid hydrogel particles, i.e., they combine the size distribution and internal structural traits found

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Biochemistry

Figure 6. Flow cytometry 2-D density plots (FSC vs SSC) of 5 µM r-n16.3, r-Pif97, and 1:1 r-n16.3 : r-Pif97 samples in 10

mM HEPES, pH 8.0 (apo) and 10 mM HEPES, 10 mM CaCl2, pH 8.0. FSC meaures light scattered less than 10 degrees as a particle passes through the laser beam and is related to particle size. SSC is a measurement of mostly refracted and reflected light that occurs at any interface within the within the particle where there is a change in refractive index. SSC is collected at approximately 90 degrees to the laser beam by a collection lens and then redirected by a beam splitter to the appropriate detector.38-42 Note that in some samples particles whose sizes fall below the 5 µm threshold are subjected to signal amplification by the instrument, leading to their display as a continuum of points along both axes; this sensitivity/resolution is described in the Methods section.

in both individual proteins. Without Ca2+, there is no apparent hybrid hydrogel formation. Evidence for selective r-n16.3 – r-Pif97 interactions. The formation of hybrid hydrogel particles in Ca2+ buffered media can only occur if there are Ca2+ - promoted interactions between r-n16.3 and r-Pif97 in solution. To ascertain this possibility, we conducted QCM-D28,29,43 to probe for non-covalent interactions between the two nacre framework proteins. Two scenarios were examined: 1) 10 mM HEPES, pH 8.0, and 2) 10 mM HEPES, 10 mM CaCl2, pH 8.0, to mimic mineralization conditions without interference from ACC or calcite precipitation (Figure 7). In scenario (2) Ca2+ ions will promote protein aggregation and thus pre-aggregation of the flow-introduced protein component will occur prior to interaction with the surface-adsorbed protein component.28,29 In these studies, poly(L-lysine)-coated Au QCM-D sensors28,29,43 were utilized to provide a biocompatible surface for initial r-n16.3 protein adsorption [note

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that both r-n16.3 and r-Pif97 contain Asp and Glu residues and thus should be electrostatically compatible with poly(L-lysine) coatings].

Figure 7. QCM-D experiments of immobilized r-n16.3 (2.5 μM in HEPES) exposed to 2.5 μM r-Pif97 under two different conditions: rPif97 in 10 mM HEPES (pH 8.0) or r-Pif97 in 10 mM HEPES / 10 mM CaCl2 (pH 8.0). Plots show the fifth harmonic frequency (F5, blue) and dissipation (D5, red) observed under each scenario. The time-dependent introduction of r-Pif97 alone (circles) or r-Pif97+Ca2+ (lines) and HEPES washing solutions is noted on the plots by arrows and extended dashed lines. These experiments were duplicated (see Figure S3) and found to be reproducible.

To probe for protein-protein interactions, we first created a layer of adsorbed r-n16.3 protein on poly(L-lysine) using a 2.5 μM protein solution in 10 mM HEPES buffer (Figure 7, first arrow). Next, we introduced r-Pif97 protein (2.5 μM) in HEPES buffer or 10 mM CaCl2 solution in HEPES buffer (Figure 7, second arrow) onto this immobilized r-n16.3 layer. In the absence of Ca2+, the frequency and dissipation of the functionalized sensor (which reflect mass deposition and viscoelasticity, respectively) exhibit negligible shifts upon the introduction of the second protein (Figure 7). This indicates that under apo conditions the two framework proteins do not interact appreciably. However, when Ca2+ and r-Pif97 are both present in the running buffer, we note a decrease in frequency, corresponding to mass deposition, as well as a considerable increase in the dissipation and

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Biochemistry

thus the viscoelasticity of the protein layer on the sensor surface. Both frequency and dissipation only slightly change when purging Ca2+out of the flowcell with 10 mM HEPES buffer alone (Figure 7, third arrow). These results were reproducible (Figure S3, Supporting Information) and indicate that, although the two proteins do not significantly interact within a Ca2+ - free buffered medium at pH 8.0, the addition of Ca2+ ions promote non-covalent interactions between r-Pif97 and the r-n16.3 layer. Based on deposited mass and molecular weight of the proteins, using the Sauerbrey equation we estimate that in the presence of Ca2+ there are 4.7x1012 r-n16.3 proteins adsorbed per cm2 compared to 1.3x1012 r-Pif97 per cm2, or 3.6 r-n16.3 molecules for every 1 r-Pif97 molecule, i.e. ~ a 4:1 binding ratio. However, we note that the Sauerbrey equation assumes a rigid layer with low viscoelasticity. In the presence of Ca2+ we observe a marked increase in the dissipation upon exposure of surface immobilized r-n16.3 to r-Pif97 (Figure 7) and this increase in dissipation is characteristic of a viscoelastic layer. Thus, in our experiments the Sauerbrey equation probably underestimates the immobilized mass of r-Pif97 and we conclude that the 4:1 ratio represents an estimate. Nonetheless, these findings imply that there are multiple r-n16.3 binding sites on the r-Pif97 protein. From Figure 1, we note that both proteins possess short sequence clusters of anionic residues, Asp and Glu, which represent putative sites for Ca2+ binding, and hence potential bridging sites for Ca2+ - mediated protein-protein interactions.29 To verify our results obtained with Ca2+, we ran parallel experiments where protein-deficient HEPES/Ca2+ buffer was injected over the r-n16.3 functionalized sensor (Figure S4, Supporting Information). In this instance, although initial shifts in frequency and dissipation were observed, upon washing with HEPES the final mass and viscoelasticity increase was comparably less than that of the HEPES/Ca2+ + r-Pif97 scenario. Also, the two curves followed different kinetics, where that of HEPES/Ca2+ alone resembled less that of a typical binding isotherm. Thus, during the mineralization assay, Ca2+ ions promote r-Pif97 – r-n16.3 interactions (Figure 7), which leads to the formation of hybrid

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hydrogel phases or particles (Figure 6). It is presumed that these hybrid hydrogel particles are responsible for observed crystal surface nanotexturing (Figure 2) and non-classical nucleation kinetics and ACC solubilities (Figures 4, 5, Table 1).

DISCUSSION There are numerous beta-chitin associated framework proteins found in the pearl oyster nacre layer and their expression and co-appearance during shell formation suggests that certain proteins may work together in the nucleation and formation of fracture-toughened mesocrystal aragonite tablets.21,23,24 In this present study, we discovered that the recombinant forms of two P. fucata framework proteins, n16.38-13,17-22 and Pif97,8-12,14,23-27 in a 1:1 molar ratio, form hybrid hydrogels that exhibit selective synergistic functionalities on specific stages or phenomena that occur during calcium carbonate nucleation. Regarding crystal formation (Figures 2, 3), there appears to be little if any evidence of synergistic activity: the hydrogel phase formed by the 1:1 r-n16.3 : r-Pif97 combination essentially allows each protein to perform its specific task vis a vis nanotexturing, alteration in growth direction, and intracrystalline incorporation into the forming crystal. However, during the early phase of nucleation, we saw a selective synergistic effect when both proteins are present: enhanced inhibition of nucleation (Figure 4A, 5A, Table 1) and the initial stabilization of transient or more soluble ACC phases that eventually transform into a less soluble phase (Figure 5B, Table 1). Mechanistically, we speculate that this r-n16.3 – r-Pif97 synergism occurs via “smart” hydrogel binding and release of water,39 leading to the inhibition of the dehydration processes required for dense mineral droplet formation directly from solute PNC precursors upon phase separation, as well as for the formation of solid ACC from the liquid intermediates.37 In contrast, a different synergism scenario was observed between the two recombinant P. fucata pearl-associated nacre proteins, r-PFMG1 and r-PFMG2, wherein the two proteins exhibited

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cooperativity during the later stages of crystal growth, but during the early stages of non-classical nucleation only r-PFMG1 was found to be influential.29 Thus, different nacre protein pairs exhibit different degrees or modes of synergism within in vitro calcium carbonate environments. From these results, we speculate that within the nacre layer specific combinations of proteins will elicit specific mineralization functionalities that manage certain stages of nacre formation. But what is the molecular basis for the functional synergism that we observe with the r-n16.3 – r-Pif97 combination? It appears to be Ca2+ - mediated interactions between the two proteins (Figure 7). Previous studies documented the enhanced hydrogelation response that both proteins exhibit in the presence of Ca2+, which implies that both proteins are Ca2+ -interactive.19,25 Very rough calculations from our QCM-D data suggest that the approximately 4 molecules of r-n16.3 complex with every 1 molecule of r-Pif97. This suggests that there are multiple r-n16.3 interaction sites on the r-Pif97 sequence that become active when Ca2+ is present. This molecular complexation, in turn, leads to the formation of hybrid hydrogel particles (Figure 6) that are now more nucleation-responsive (Figures 4,5, Table 1). A similar Ca2+ - mediated interaction was noted for the r-PFMG1 – r-PFMG2 pearl-associated protein pair,29 and in both cases protein-protein interactions would be triggered within the Ca2+ / CO3-2 / HCO3nucleation environment of the pearl and shell nacre layers. Analysis of the primary sequences for both framework proteins (Figure 1) clearly shows that there are anionic Asp and Glu short sequence clusters found in both the n16.3 and Pif97 proteins, particularly within the Pif97 vWA domain (F23-Y161) and the EGF-like domain of n16.3 (D27 – L60). These Asp and Glu sites could serve at least two potential functions in situ: 1) trigger protein-protein interaction (Figure 7) leading to hydrogel assembly (Figure 6) and 2) regulate the nucleation process (Figures 4, 5). Thus, we speculate that the vWA and EGF-like motifs may represent putative hydrogelation and mineralization sites within Pif97 and n16.3, respectively. Obviously, this expands our understanding of the functional capabilities of vWA and EGF-like sequences

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within extracellular processes such as biomineralization. Lastly, we would like to place our in vitro observations of the combinatorial n16.3 – Pif97 hydrogel complexes within the context of nacre layer development1-7 and the potential role(s) that multiple framework proteins8-29 might play in this process. Given that the mesoscale nacre aragonite tables form from the assembly of nanoparticle precursors (presumably ACC),4-7 it is plausible that hybrid framework protein complexes or hydrogels could regulate the kinetics and solubilities of these precursor clusters (Figures 4, 5, Table 1). The fact that Pif97 has been established to be a calcite-inhibitor protein2327

and n16.3 an aragonite stabilizer/organizer and calcite-inhibitor, by combining these two proteins into

a hybrid hydrogel network the mollusk could achieve more effective control over ACC formation/stabilization/transformation events and thereby block calcite formation (Figures 4, 5). This effect could multiply even further if other framework proteins were to participate in multi-protein hybrid hydrogel formation. If the thermodynamically more stable calcite polymorph cannot form, then the metastable aragonite polymorph, assisted by Mg2+ ions,17,24,44 may be stabilized as nanoparticles by the multi-protein hydrogel17,19 and assemble in place of calcite at the framework organic layer boundary. Furthermore, as the formation of the crystalline phase proceeds, these same multi-protein hydrogels would occlude within the growing crystals over time (Figure 3), thus establishing intracrystalline nanoporosities which are critical for fracture toughness.1-7 This type of combinatorial control can be further amplified and augmented by the presence of other nacre proteins that could form complexes with n16.3 – Pif97, such as Pif80, a putative aragonite-promoting protein of the P. fucata framework proteome.23,24,26,27 Obviously, additional in vitro experiments coupled with in situ probing of nacre formation will be required to fully establish the process of framework protein management of mesoscale aragonite tablets from nanoparticles.

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ASSOCIATED CONTENT Supporting Information. Enlargement of SEM image showing representative calcite crystals generated in 1:1 r-n16.3 : r-Pif97 combinatorial mineralization assay (Figure S1); Experimental procedures, MicroRaman spectra and corresponding light microscopy images of protein-deficient, r-n16.3, r-Pif97, and 1:1 r-n16.3 : r-Pif97 combinatorial mineralization assays (Figure S2, Table S1); Reproducibility of QCM-D measurements (Figure S3); Control QCM-D experiment to test the effect of Ca(II) on the rn16.3 adsorbed layer (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Dr. Gaurav Jain

[email protected]

Dr. Martin Pendola

[email protected]

Dr. John Spencer Evans

[email protected]

Yu-Chieh Huang

[email protected]

Denis Gebauer

[email protected]

Eleni Koutsoumpeli

[email protected]

Steven Johnson

[email protected]

Corresponding Author *To whom correspondence should be addressed: John Spencer Evans, Laboratory for Chemical Physics, Division of Basic Sciences and Center for Skeletal and Craniofacial Medicine, New York University College of Dentistry, 345 E. 24th Street, New York, NY, 10010. Tel.: (212) 998-9605; Fax: (212) 9954087. Email: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Portions of this research (protein production and purification, assays, SEM, AFM) were supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award DE-FG02-03ER46099 (JSE), and the QCM-D research was supported by grants from the UK Engineering and Physical Sciences Research Council (EP/M02757/1 and EP/P030017/1)(EK, SJ)

ACKNOWLEDGMENT

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This paper represents Contribution Number 91 from the Laboratory for Chemical Physics, New York University.

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For Table of Contents use only

Selective synergism created by interactive nacre framework-associated n16.3 and Pif97 proteins: Implications for nacre layer formation.

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Gaurav Jain, Martin Pendola, Yu-Chieh Huang, Denis Gebauer, Eleni Koutsoumpeli, Steven Johnson, and John Spencer Evans

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