Insights into Mollusk Shell Formation: Interlamellar and Lamellar

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Insights into Mollusk Shell Formation: Interlamellar and Lamellarspecific Nacre Protein Hydrogels Differ in Ion Interaction Signatures Martin Pendola, and John Spencer Evans J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10915 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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The Journal of Physical Chemistry

Insights into Mollusk Shell Formation: Interlamellar and Lamellar-specific Nacre Protein Hydrogels Differ in Ion Interaction Signatures

Martin Pendola and John Spencer Evans*

Laboratory for Chemical Physics, Center for Skeletal and Craniofacial Biology, New York University, 345 E. 24th Street, NY, NY, 10010 U.S.A. *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) 995-4087. Email: [email protected].

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ABSTRACT. In the mollusk shell nacre layer there exist hydrogelator proteomes that play important roles in the formation of the mineral phase.

Two of these proteomes, the

intracrystalline and the framework, reside in the interior and exterior, respectively, of the nacre tablets. To date there is no clear evidence of what distinguishes an intracrystalline protein from a framework protein regarding the nucleation process.

Using Eu(III), phosphate anions, and

recombinant versions of the intracrystalline protein, AP7 and the framework protein, n16.3 we probed each protein hydrogel for its interactions with these model ions.

Fluorescence

spectroscopy of Eu(III) interactions with both protein hydrogels revealed that r-AP7 exhibited enhanced effects on Eu(III) fluorescence compared to r-n16.3, and,

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P NMR experiments

demonstrated that r-AP7 had a more significant impact on phosphate anions compared to rn16.3. Thus, r-AP7 was found to be more of an ion “disruptor” than r-n16.3. Interestingly, these findings correlate with the particle size distributions and internal structure of the hydrogel particles themselves, suggesting that the physical properties of the hydrogels dictate hydrogel-ion interactions. In conclusion, we confirm that hydrogelator proteomes possess distinguishable ion interaction properties that may impact the nucleation processes in these regions and control the overall formation of mesoscale nacre tablets.


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INTRODUCTION Biological organisms offer many potential models where multiple material properties and construction phenomena intertwine at the mesoscale. A case in point is biomineralization, a process by which organisms employ assembly mechanisms to create inorganic-organic skeletal elements over different length scales (nano to macro).1-9 In some biomineralizing systems, such as the invertebrate mollusk, the nacre layer of the shell consists of organized mesoscale single crystal aragonite tablets.1-10

These mesoscale tablets were formed from the assembly of

amorphous calcium carbonate (ACC) nanoparticles during the development of the shell.11,12 This assembly process is believed to be guided by nacre-associated proteins that reside in the nacre layer.13-19 These shell-specific proteins are hydrogelators20-25 and occur in 2 classes:13-19 a) Intracrystalline, historically referred to as the “acid-soluble” protein family, which reside within the interlamellar region of the nacre tablets; b) Framework, historically referred to as the “acid-insoluble” protein family which co-exist with β-chitin polysaccharide films and silk proteins that comprise the lamellar layers that surround each tablet. Although it is not clear why separate protein families or proteomes are required for nacre formation, past studies have shown that members of each class differ in their in vitro mineralization properties, such as ACC formation and stabilization, surface modifications to crystal surfaces and the location and size of intracrystalline porosities, and the organization of mineral nanoparticles within the hydrogel matrices.20-24 This suggests that members of each protein class differentially manage the nucleation and crystal assembly processes in the mollusk shell. We speculate that the differences in nacre proteomes may lie at the level of hydrogel matrix properties and how these matrices manage or interact with ions during the nucleation process.20,24 To test this idea, we performed parallel experiments on recombinant variants of two 3 ACS Paragon Plus Environment


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nacre-associated proteins, AP725 (r-AP7,26,27 Haliotis rufescens, intracrystalline class, MW = 7.5 kDa; pI = 5.43; net protein charge @ pH 8.0 = -3.5) and n16.328 (r-n16.3,20,23 Pinctada fucata, framework class, MW = 12.9 kDa; pI = 4.82; net protein charge @ pH 8.0 = -7.8) where we examined the ability of each nacre protein hydrogel to form clusters with multivalent cations [i.e., Eu(III),29-31 a fluorescent Lanthanide ion that acts as a surrogate for Ca(II)]32,33 and interact with anions (i.e., Na3PO4, a surrogate for carbonate/bicarbonate ions that avoids carbon dioxide outgassing and pH shift issues)34 at pH 8.0, the relevant condition for in vitro calcium carbonate nucleation in the presence of nacre proteins.20-24 We discovered that each nacre protein hydrogel exhibits a different “signature” or profile of metal ion complexation and phosphate ion diffusion: there are stoichiometric-dependent differences in the interactions of r-AP7 and r-n16.3 hydrogels with Eu(III) ions, and, protein-dependent differences in phosphate ion diffusion.

A flow

cytometry examination of the protein hydrogel particles in the presence of Eu(III) cations and phosphate anions reveals that the particle size distributions and internal structure of r-AP7 and rn16.3 hydrogel particles correlate with their respective ionic “signatures”.

Thus, protein

hydrogelators which reside in different regions of the forming nacre layer possess distinguishable cation and anion interaction properties that are linked to their hydrogel particle properties. The differences in these properties may explain why each nacre protein hydrogel exhibits different effects on ACC formation and crystal morphologies in vitro,20,23,26,27 and may explain the need for regional protein families within different ion interaction signatures to control the overall formation of mesoscale nacre tablets.1-10

METHODS


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Recombinant Expression and Purification of Nacre Proteins. The bacterial expression and purification of recombinant AP7 (r-AP7) and n16.3 (r-n16.3) were performed as described in earlier studies.25,26 Both proteins were stored as lyophilized solids at -80 oC until needed, and stock solutions of each protein were created using 30 nm filtered ultrapure molecular biology grade water (Fisher Scientific, U.S.A.). Eu(III) Titrations of Nacre Protein Hydrogels.

To probe the interactions of

multivalent cations with nacre protein hydrogels, we relied on the Ca(II) surrogate, Eu(III),32,33 and fluorescence spectroscopy.29-33 For titrations, 1 M EuCl3 (99.9% pure, Alfa Aesar, Inc) stock solutions were generated using 30 nm filtered molecular biology grade water (Fisher Scientific, U.S.A.). In control scenarios (i.e., no protein), EuCl3 samples of 10, 20, 50, 100, 150, 200 µM in 10 mM HEPES, pH 8.0 (i.e., the relevant calcium carbonate mineralization pH) were created. For protein scenarios, parallel EuCl3 samples containing 10 µM r-AP7 or r-n16.3 in 10 mM HEPES, pH 8.0, were created, leading to solutions with protein: Eu(III) molar stoichiometries of 1:1, 1:2, 1:5, 1:10, 1:20.

All samples (in triplicate) were monitored for their intrinsic

fluorescence at 550 to 750 nm using a Horiba PTI Quanta Master 400 Fluorescence Spectrometer using an excitation wavelength of 390 nm at 25 oC, with buffer background subtraction. 2-D

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P-NMR DOSY Diffusion Experiments. Since carbonate/bicarbonate solutions

are prone to carbon dioxide outgassing and subsequent pH shifts,34 we chose phosphate anion as a surrogate for the carbonate/bicarbonate species for measurements of bulk solution anion diffusion in the presence of nacre protein hydrogels. Samples for NMR diffusion measurements were prepared using Na3PO4 (98+%, Acros Chemicals, U.S.A.) at 1 mM concentration (pH 8.0) in 90% v/v 30 nm filtered molecular biology grade water (Fisher Scientific, U.S.A.) and 10% v/v D20 (99.9% atom D, Cambridge Isotopes Lab, U.S.A.) in a final volume of 150 µL. The control



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sample contained no added protein, and the r-AP7, r-n16.3 final concentrations were 50 µM each.

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P NMR 1-D and 2-D DOSY diffusion-ordered spectroscopy experiments35-39 were

conducted on bulk solution phosphate anions using a Bruker AVANCE-600 MHz spectrometer system at the New York Structural Biology Center (NYSBC) using a TPI cryoprobe and 3mm NMR tubes. Acquisition parameters for the 1D experiments included 2400 scans, a recovery delay of 10 sec, and a data size of 8K). For the 2D DOSY experiments, the acquisition parameters included 10 scans/increment, recovery delay 10 sec, linear gradient 2-98%, F1 spectral width = 10.01 ppm, F2 spectral width = 39.6 ppm) at 25 oC. were referenced from external 85% H3PO4

40

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P NMR chemical shifts

and NMR spectra were generated using Bruker

TopSpin software.39 Flow Cytometry Studies of Nacre Protein Hydrogel Particles. The physical state of rAP7 and r-n16.3 protein hydrogel particles were studied in parallel under the following conditions: a) 1 mM Na3PO4, pH 8.0; b) 10 mM HEPES 100 µM EuCl3, pH 8.0, and c) 10 mM HEPES 200 µM EuCl3, pH 8.0. In the case of the Na3PO4 experiments, protein concentrations were 50 µM as per the NMR experiments described above, and in the case of the Eu(III) experiments, protein concentrations were 10 µM as per the fluorescent spectroscopy experiments.

Samples were constituted and allowed to sit for 5 min prior to analysis.

Aggregation measurements were performed using a multi-parameter cell analyzer BD LSRFortessa (BD Biosciences, U.S.A.) with sensitivity in the 1.5 – 2.0 µm range and resolution within the 5-20 µm range for both FSC and SSC parameters. Each sample solution (150 µL) was analyzed at a continuous flow rate of 25 µL/min using four laser excitation lines 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


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found in the legend to Figures 5,6)39,41-43 and the number of events for each sample. Data was collected using the BD FACS DiVa software (BD Biosciences, U.S.A.) designed for the instrument and processed using FlowJo software (TreeStar, OR, U.S.A.). For the flow cytometry data presented in Figures 5 and 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 that form in the presence of Eu(III) and phosphate ions, and are detected and compensated via signal amplification by the instrument. This compensation leads to the continuous display of datapoints along the edges of a given chart due to the presence of non-resolvable small protein particles in the sample.

RESULTS AND DISCUSSION The in vitro non-classical nucleation process involves the formation of ionic nanoclusters of Ca(II), HCO3-, CO32-, known as pre-nucleation clusters (PNCs)44-47 and their assembly into the precursor phase of nacre aragonite, amorphous calcium carbonate (ACC), via particle attachment.48,49 As summarized in previous studies,20,23,26,27 both r-AP7 and r-n16.3 hydrogels are nearly equivalent in their ability to extend the time interval of the in vitro non-classical ACC nucleation process (Figure 1A).44-47 However, there are distinctive protein-specific differences for other aspects of the non-classical nucleation process.

Specifically, PNC stabilities, as

measured by the pre-nucleation slope of the Ca(II) titration curve (Fig 1B),44-47 are quite distinct for each protein, as are ACC solubilities (Figure 1C), with r-n16.3 exhibiting a higher degree of ACC stabilization and r-AP7 neither stabilizing nor destabilizing ACC clusters.44-47 Moreover, the effects that each protein hydrogel exert on the growth of existing calcite crystals is quite different: When r-AP7 hydrogels contact crystal surfaces, they inhibit crystal growth and create 7 ACS Paragon Plus Environment


Figure 1. Summary of non-classical Ca(II) potentiometric titrations of AP7 and n16.3 hydrogels at pH 9.0. A) Nucleation time; B) PNC stability (taken from the slope of the titration curve); C). ACC solubility. Reference values (i.e., protein-free controls) were subtracted from all protein values. D) SEM images of calcite crystals generated by each protein (50 µM) in 60 min mineralization assays (pH 8.0). All data were taken from references 22,23,26. highly ordered nanotextured surfaces on calcite.50 Conversely, under the same conditions crystal surface-adsorbed r-n16.3 hydrogels introduce both nanotexturing and new growth directions (Figure 1D).51 Given that both proteins manipulate the nucleation and crystal growth processes in different ways, we were interested in the ability of each nacre protein hydrogel to interact with bulk solution cations and anions, since these types of interactions could regulate ion availability for PNC and ACC formation as well as ion deposition on crystal surfaces. Nacre Protein Hydrogels Exhibit Different Cation Interaction Signatures. To examine cation-hydrogel interactions we performed stoichiometric Eu(III) titrations of 10 µM rAP7 and r-n16.3 samples in 10 mM HEPES buffer, pH 8.0, and monitored changes in Eu(III) intrinsic fluorescence intensities and wavelengths (Figures 2,3). Note that Eu(III) is being utilized here as a Ca2+ ion surrogate32,33 and that pH 8.0 represents the in vitro mineralization assay pH conditions that were reported in earlier nacre protein hydrogel studies;20,23,26,27 8 ACS Paragon Plus Environment

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unfortunately, the in-situ pH of the nacre layer during mineral formation is not known and thus we must default to in vitro values. Compared to the protein-free state (i.e., the negative control), both r-AP7 and r-n16.3 possess the same number of Eu(III) emission bands and experience significant increases in fluorescence intensities for the expected signature bands at 590 nm (5D0 → 7F1 transition) and 616 nm (5D0 → 7F2 transition) as well as the minor emission band at 697 nm (5D0 → 7F4 transition)29-31 (Figure 2).

Figure 2. Fluorescence emission spectra of 200 µM EuCl3 in the presence and absence of 10 µM nacre protein hydrogels in 10 mM HEPES, pH 8.0, i.e., 20:1 Eu(III): protein molar ratio Note that no protein-induced wavelength shifts were observed for these transitions. As noted in seminal Eu(III) fluorescence studies,29-31 the formation of a unique asymmetric environment surrounding an Eu(III) ion leads to a marked enhancement of its fluorescence relative to the control scenario, primarily due to the increased transition probability for fluorescence but also due the sensitivity of the first solvation sphere of the Eu(III) ion, which becomes perturbed when interactions occur between Eu(III) and chelators such as protein-associated Asp and Glu carboxylate groups.32,33 9 ACS Paragon Plus Environment


As shown in Figure 3, a plot of the 590 nm, 616 nm, and 697 nm fluorescence intensities as a function of Eu(III) : protein stoichiometries (1:1, 1:2, 1:5, 1:10, 1:20) reveals a general trend towards higher fluorescence intensities for both r-AP7 and r-n16.3 relative to the control. We believe that general trend in increasing fluorescence intensity for both proteins reflect the following phenomena: 1) Eu(III) introduction leads to asymmetric interactions of Eu(III) ions with Asp and Glu sites,32,33 which enhances Eu(III) fluorescence, and 2) as Eu(III) concentration increases, so does hydrogelation,20,23,26,27 and this induces environmental changes for proteininteracting Eu(III) ions which further enhances Eu(III) fluorescence intensity.


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Figure 3. Plot of 590 nm, 616 nm, and 697 nm fluorescence emission intensities as a function of Eu(III):protein mole stoichiometries [note that for the protein-free curve, the stoichiometries correspond to Eu(III) concentrations of 10, 20, 50, 100, 150, 200 µM]. Each data point represents triplicate samples. We now focus on the protein-specific differences in metal ion complexation. Interestingly, in the Eu(III) : protein stoichiometry range of 1:1 to 10:1, Eu(III) fluorescence enhancement follows the relationship r-n16.3 > r-AP7 > control, and is more pronounced for rn16.3 hydrogels than r-AP7 hydrogels for all three transition bands (Figure 3). At 20:1 r-AP7 exhibits slightly higher fluorescence intensities and r-n16.3 exhibits a notable drop in fluorescence intensity. This level of carboxylate – cation binding and/or environmental change compared to r-AP7, perhaps reflecting the differences in Asp and Glu content in each protein (rn16.3 = 18 Asp+Glu, net protein charge @ pH 8.0 = -7.8; r-AP7 = 8 Asp+Glu, net protein charge @ pH 8.0 = -3.5).25,28 However, at > 10:1 stoichiometries, r-n16.3 Eu(III) fluorescence emission decreases as r-AP7 fluorescence enhancement continues to increase (Figure 3). Thus at > 10:1 ratios these protein-specific effects on fluorescence intensities cannot be explained by the number of protein-associated carboxylate groups or net protein charge alone, but must reflect other differences that exist, which we will discuss below. In conclusion, although r-AP7 and rn16.3 hydrogels both interact with Ca2+ surrogate Eu(III) ions, each protein hydrogel exhibits a different cation interaction signature that is stoichiometric-dependent. Signature Hydrogel-Phosphate Interactions Occur in the Presence of r-AP7 and rn16.3. Our next step was to assess r-AP7 and r-n16.3 protein hydrogel – bulk anion interaction signatures, since this would be relevant for interactions with anionic carbonate/bicarbonate molecules during the early stages of the nucleation process.43-46 11 ACS Paragon Plus Environment

Ideally, we would have


performed this assessment with carbonate/bicarbonate anions, which are relevant to the nacre mineralization process. However, we found that carbon dioxide outgassing34 occurred over time, leading to pH shifts that were unacceptable. To circumvent this problem, we employed sodium phosphate, Na3(PO4) as a pH-stable surrogate for the carbonate/bicarbonate species, and utilized 31

P NMR 1-D as well as 2-D DOSY or Diffusion Ordered SpectroscopY35-39 experiments to

determine the diffusion coefficients for 1 mM phosphate solutions (pH 8.0) that were either protein-free or contained 50 µM of either protein hydrogel (a 20:1 phosphate : protein molar ratio). As described in our previous 1-H NMR DOSY studies with sea urchin protein hydrogels,39 in the

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P 2-D DOSY experiment the phosphate groups become spatially labeled

based on their position in the NMR tube. If these molecules move during the encoding, or ‘labeling’, period within the diffusion interval (Δ) that follows in the pulse sequence, their new position can be decoded with a second gradient (F1 axis) and a temperature-dependent diffusion coefficient can be calculated.35-39 Thus, any phosphate molecules which interact with a hydrogel particle ensemble should exhibit a noticeable change in their diffusion coefficients relative to


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Figure 4.

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P NMR spectra of 1 mM Na3PO4 in 10% v/v D2O/90% 30nm filtered ultrapure

water, pH 8.0, in the presence and absence (control) of 50 µM nacre protein hydrogels.

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P

NMR chemical shifts referenced from external 85% v/v H3PO4 free phosphate species in the absence of protein. Three caveats should be mentioned: (1) We presume that the large, denser hydrogel particles have settled out to the bottom of the tube during the NMR experiments; however, there may be some population of hydrogel particles whose sizes and densities allow them to remain suspended in the solution rather than settle out. Therefore, the dataset reflects both hydrogel states and their averaged effects on bulk phosphate anion diffusion.39 (2) We assume that hydrogel-induced perturbations with phosphate molecules reflect averaging of internal and external hydrogel surface interactions with the phosphate anions, which unfortunately our NMR experiments are unable to resolve as separate phenomena.39 3) Since DOSY experiments are optimized for isotropic species,35-39 any phosphate molecules that are tightly bound to hydrogel particles becomes anisotropic and thus cannot be detected by these experiments due to inhomogeneous broadening effects. Thus, the DOSY experiments primarily measure the effect of each hydrogel on the diffusion of mobile, exchangeable bulk phosphate species.39 As shown in Figure 4 and Table 1, the 31P NMR 1-D spectra for phosphate anions in the presence of r-AP7 and r-n16.3 reveals a hydrogel-induced downfield chemical shift effect (+0.45 ppm, r-AP7; +1.75 ppm, r-n16.3) relative to the 85% v/v H3PO4 reference standard. Note that relative to the control sample, there is a downfield 31P NMR chemical shifts for phosphate anions in the presence of r-n16.3, but an upfield shift is observed for phosphate anions in the presence of r-AP7. This indicates that the phosphate anions experience hydrogel induced perturbations (e.g., changes in phosphate group electronic distribution, bond angles, dynamics) that influence 13 ACS Paragon Plus Environment


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the 31P NMR chemical shift of these molecules in the presence of each protein hydrogel sample. These results also correlate with proportional to the

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P linewidths at half-height (∆ν1/2) which are inversely

P phosphate spin-spin relaxation time, T2. Here, ∆ν1/2 = 44 Hz for the

control sample, and 13 Hz and 15 Hz for the r-AP7 and r-n16.3 samples, respectively, ~ 2-3-fold difference from the control values. Collectively, the 1-D NMR dataset reveals that the bulk solution phosphate anions are interacting and exchanging with both protein hydrogel particles.3539

However, the protein-specific differences in

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P NMR chemical shifts relative to the control

sample (r-AP7 = -0.96 ppm; r-n16.3 = 0.34 ppm) suggest that the interactions, behavior, and exchange of the bulk phosphate anions with r-AP7 hydrogels are different. As we shall see below, we believe that this difference has to do with the presence of different of r-AP7 hydrogel particles at pH 8.0. These conclusions are also supported by the 2D-DOSY spectra (Figure 5).

The

calculated diffusion coefficients (Table 1) clearly show that both hydrogels perturb the diffusion of bulk phosphate anions, with D25, AP7 and D25, n16.3 values being ~10 - 100x larger than D25, control.

Diffusion coefficients which exceed those of the control scenario suggest that both

protein hydrogels are rapidly interacting with the bulk phosphate anions, causing them to diffuse at a faster rate compared to the control sample.35-39 However, a closer examination of the DOSY dataset reveals that there are differences in phosphate diffusion rates for the r-n16.3 and r-AP7 samples (Figure 5, Table 1). The r-n16.3 sample exhibits a single phosphate resonance with a diffusion coefficient that is ~100x higher than the control value. In contrast, the AP7 sample is more different (i.e., Peak 1 : Peak 2 intensity ratio ~ 2). The fact that both r-AP7-associated phosphate


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Figure 5. 3D 31P NMR DOSY spectra of 1 mM Na3PO4 in the presence and absence of 50 µM nacre protein samples. The x-axis (F2) provides the 31P NMR chemical shift (in ppm) the y-axis (F1) provides the log of the diffusion coefficient (m2/sec) at 25 oC, and the z-axis represents the spin population for each peak. The individual r-AP7 peaks are labeled.

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P NMR chemical

shifts are referenced from external 85% v/v H3PO4. diffusion coefficients are different from that of the control (Table 1) indicates that the 31P species represented by Peaks 1 and 2 are interacting with r-AP7, i.e., neither represents unperturbed phosphate molecules. Moreover, the diffusion coefficient obtained for Peak 1 represents the largest perturbation from the control value (Table 1). Potentially, there are two plausible explanations for Peaks 1,2 in the r-AP7 DOSY spectra. 1) There may be two sites of interaction for phosphate groups on all r-AP7 hydrogel particles, which each site having a different binding constant and thus a different diffusion rate for each site. 2) There exist two distinct r-AP7 hydrogel particle populations, each with its own phosphate interaction signature. As we describe below, it appears that the latter scenario may be at play here. Table 1: Sample

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P NMR parameters obtained for1 mM sodium phosphate (pH 8.0) in the presence and absence of 50 µM nacre protein hydrogels. 31 1-D 31P NMR Chemical Diffusion Coefficient (D, 25 P ∆ν1/2 o Shift (ppm) C, m2/sec) (Hz)

Control (no protein) r-AP7

1.41

44

1.84 x 10-11

0.45

13

r-n16.3

1.75

15

Peak 1: 2.38 x 10-10 Peak 2: 3.16 x 10-9 1.78 x 10-9



Correlation between Hydrogel-Ion Interactions and Hydrogel Particle Parameters. Previous studies noted that there are pH and Ca(II)-dependent differences in nacre protein hydrogel particle size distributions and internal structure.20,24,27,39 Hypothetically, both Eu(III) and phosphate ion interactions could influence nacre protein hydrogels in unexpected ways, and thus we were interested in determining how our experimental conditions affected protein hydrogel particles. To achieve this, we undertook a flow cytometry39,41-43 study of the two hydrogels under the following conditions: a) 10:1 and 20:1 Eu(III) : protein stoichiometry points, where we noted significant fluorescence intensity differences between the two hydrogels (Figure 3), and b) 1 mM Na3PO4, pH 8.0 (Figure 5). Note that in flow cytometry there are two light scattering parameters that one can monitor for particles under constant flow: 1) forward scattered light component (FSC, x-axis) to determine particle size distribution (note that our flow cytometry experiments do not provide exact particle size data, only distributions of particle sizes);

39,41-43

and 2) side-scattered light component (SSC, y-axis) to measure refracted and

reflected light that occurs at any interface within the particles where there is a change in refractive index (RI) that results from variations in particle granularity or internal structure (further information can be found in the legend to Figure 6). 39,41-43 As shown in Figure 6, hydrogel particle parameters correlate with hydrogel – Eu(III) ion interaction signatures. A comparison of r-AP7 versus r-n16.3 hydrogels at 10:1 and 20:1 Eu(III) : protein stoichiometries reveal that the r-AP7 hydrogel particles experience ~ 50% reduction in particle size distribution and, most notably, ~ 150% increase in internal particle granularity or structure. We interpret this result as follows: As Eu(III) ions interact with r-AP7 hydrogel particles, these cations induce structural or organizational changes within or between hydrogel


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particles (Figure 6A,B) that somehow facilitate additional Eu(III) – r-AP7 hydrogel interactions, which would contribute to the increase in Eu(III) fluorescence intensity noted over this stoichiometric range (Figure 3).

In contrast, r-n16.3 hydrogel particles experience ~ 50%

increase in particle size distributions but exhibit minimal change in particle granularity or structure (Figure 6C, D). This lack of Eu(III) – induced r-n16.3 hydrogel internal structural or organizational change correlates with the reduction in Eu(III) fluorescence intensities noted in the 10:1 to 20:1 Eu(III):protein ratio range (Figure 3), and indicates that Eu(III) may not be inducing sufficient structural changes to r-n16.3 hydrogel particles that would lead to additional ion binding.

Figure 6: Flow cytometry 2-D density plots (FSC vs SSC) of 10 µM r-AP7 and r-n16.3 hydrogel particles under the following conditions. (A) 10:1 Eu(III):r-AP7; (B) 20:1 Eu(III):rAP7; (C) 10:1 Eu(III):r-n16.3; (D) 20:1 Eu(III):r-n16.3, all in 10 mM HEPES, pH 8.0. FSC



refers to a parameter measuring light scattered less than 10 degrees as a particle passes through the laser beam and is related to particle size. SSC is proportional to particle granularity or internal complexity and 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. Note that 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. The flow cytometry data for phosphate anions (Figure 7) also indicates that nacre protein hydrogel particle size distributions and internal granularity correlate with hydrogel – phosphate interaction “fingerprints” (Figures 4, 5). Here, we see that r-AP7 exhibits three resolvable


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Figure 7. Flow cytometry 2-D density plots (FSC vs SSC) of the r-AP7 and r-n16.3 hydrogel particles in 1 mM Na3PO4, pH 8.0. On the r-AP7 2-D density plot resolvable hydrogel particle populations are noted; note that for r-n16.3 the smaller particle populations are non-resolvable and display as a continuum along the axes. populations of hydrogel particles (labeled as 1,2,3 in Figure 6). The presence of two significant hydrogel particle populations (1,2) may explain why we detected two different diffusion-based phosphate anion populations for r-AP7 (Figure 5; Table 1). Since the third r-AP7 population (3) is minor in size, it is likely that it evades detection by 31P NMR spectroscopy and is not observed in Figures 4,5. Based upon signal intensities we suspect that Peaks 1,2 in the DOSY spectra correspond to Peaks 1,2 in the flow cytometry plot (Figures 4,7); however, further studies will probably be required to establish this with certainty. In contrast to r-AP7, r-n16.3 presents with only a single phosphate anion species (with a continuum of non-resolvable smaller particles noted along the axes limits), which explains why r-n16.3 generates a single DOSY peak (Figure 5). In summary, our flow cytometry studies confirm that the hydrogel protein particle size distributions and internal hydrogel structure contribute to the ability of a nacre protein hydrogel to interaction with bulk solution ions.

CONCLUSIONS Our present study reveals that intracrystalline r-AP7 and framework r-n16.3 protein hydrogels interact with Ca(II)- and carbonate/bicarbonate-surrogate ions in bulk solution. In the context of nucleation, hydrogel interactions with free cations and anions would affect overall ion availability, which, in turn, would interfere with the formation of ionic clusters such as PNCs and thus extend the time for nucleation, as we have previously noted (Figure 1).22,23,25 This 19 ACS Paragon Plus Environment


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competition for ions may be one basis for nacre protein hydrogel manipulations of early stages of in vitro calcium carbonate nucleation.44-47 However, although both proteins prolong ACC nucleation time, it is the r-n16.3 protein hydrogel that increases ACC stability44-47 more dramatically compared to the crystal growth inhibitor, r-AP7 (Figure 1).22,23,25 These in vitro functional differences are most likely influenced by the specific metal ion complexation and anion diffusion / availability signatures discovered for each protein (Figures 3-5, Table 1). And surprisingly, these ion interaction signatures are influenced by the hydrogel particle size distributions and internal granularity or structure of the hydrogel particles themselves (Figures 6,7). The fact that ions can affect hydrogel physical parameters and alter hydrogel-ion interactions (Figures 2-7)

20,24,27,39

adds yet another layer of complexity to the nacre protein

regulation of the in vitro non-classical nucleation process44-47 and suggests that there may be a “feedback” mechanism wherein hydrogel-ion interactions induce changes in the ability of a given hydrogel to further interact with ions (Figures 2-7). If true, then a hydrogel – ion feedback mechanism could manipulate the in vitro nucleation process in a dynamic way as available ion pools change in concentration over time. Finally, we need to consider how nacre protein hydrogel ion interaction signatures might explain events in nacre layer development. Based upon available information, it appears that the intracrystalline r-AP7 is a calcite crystal and nucleation “disruptor” in vitro (Figure 1), arising from its ion “disruptor” activity, i.e., the ability to perturb cation and anion pools (Figures 2-5, Table 1). Conversely, r-n16.3, which facilitates ACC stabilization and promotes new crystal growth directions (Figure 1), is less of an ion “disruptor”.

If we extrapolate this in vitro

relationship to the nacre layer, then we postulate that intracrystalline / interlamellar and lamellar / framework proteomes are “tuned” to interact with cations and anions in different ways (Figures


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2-5), with the protein hydrogels adjusting their particle populations and structures (Figures 6,7) in response to ion pools. This “adjustment-on-the-fly” could alter hydrogel interactions with the ions during the nucleation and crystal growth processes.

Ultimately, this may impact

mineralization events that take place within the different regions of forming mesoscale nacre tablets, such as the nucleation of ACC and its subsequent transformation to aragonite on lamellar framework beta-chitin layers (i.e., n16.3) and the inhibition of crystal formation within the interlamellar region that exists between the framework layers (i.e., AP7, the “disruptor”).52-54 These proteome-specific functionalities may ultimately control the overall formation of the aragonite nacre tablets within the mollusk shell. We await in situ studies that will confirm or refute this hypothesis.

AUTHOR INFORMATION Corresponding Authors Martin Pendola [email protected] John Spencer Evans [email protected]

Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS



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. We thank Dr. Eric Chang for his help in performing mineralization assays and SEM analyses. This report represents contribution number 89 from the Laboratory for Chemical Physics, New York University.


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