Incorporation of a Recombinant Biomineralization Fusion Protein into

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Incorporation of a Recombinant Biomineralization Fusion Protein into the Crystalline Lattice of Calcite Eva Weber,† Leonid Bloch,† Christina Guth,‡ Andy N. Fitch,§ Ingrid M. Weiss,‡ and Boaz Pokroy*,† †

Department of Materials Engineering and the Russell Berrie Nanotechnology Institute, Technion Israel Institute of Technology, Haifa 32000, Israel ‡ INM-Leibniz-Institute for New Materials, Campus D2 2, 66123 Saarbruecken, Germany § European Synchrotron Radiation Facility, B.P. 220, 38043 Grenoble Cedex, France S Supporting Information *

ABSTRACT: High-resolution synchrotron X-ray powder diffraction (XRD) combined with the Rietveld refinement method and confocal laser scanning microscopy (CLSM) were utilized in this study to elucidate the interaction between a recombinant biomineralization protein (perlucin) fused to green fluorescent protein (GFP) and synthetic calcite. Although recombinant perlucin is insoluble, its solubility was increased via fusion to the highly soluble GFP. We demonstrate that GFP-perlucin derivatives become incorporated into the calcite structure and induce concentrationdependent anisotropic lattice distortions along the host’s c-axis. In contrast, GFP alone is hardly incorporated at all. The observed lattice distortions and peculiar microstructure of the crystals are comparable to those previously observed in biogenic calcite. Taking advantage of biotechnology to optimize individual protein properties, such as the solubility of an otherwise insoluble protein derivative, is a promising route toward the synthesis of new and improved biocomposite materials.

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INTRODUCTION Biominerals demonstrate extraordinary structure−property relationships compared to their nonbiogenic counterparts.1 Organisms provide unique control not only over nucleation and the selection of crystal polymorphs but also over the orientation, microstructure, and morphology at different hierarchical levels and length scales.2−5 Deciphering the internal crystal structure of biocomposites is challenging because of the difficulty in detecting organic compounds within a crystallite structure. Moreover, the preparation of samples for characterization necessitates specific conditions owing to the combination of hard and soft matter in these biocomposite materials. Further, in order to produce artificial composites we need a good understanding of the structure and formation of the particular materials as well as specialized fabrication skills. This presupposes knowledge of the internal distribution of macromolecules. The comparison of the biogenic and the synthetic materials on the level of internal structures are of interest to mimic biological materials via chemical reactions. By far the most abundant biogenic mineral is calcium carbonate, which exists in different structural forms. Fundamental knowledge of the nano- and microstructure of biogenic calcium carbonate has been achieved by applying various synchrotron radiation methods. One such method is Xray powder diffraction (XRD). As an example, Berman et al. in 1990 reported a decrease of XRD-coherence length and the increase in peak breadth in sea urchin spines as compared to © XXXX American Chemical Society

synthetic calcite and attributed the differences to the presence of intracrystalline macromolecules in the biogenic material.6 Using high-resolution synchrotron powder XRD and the Rietveld refinement method, Pokroy and co-workers demonstrated that the structure and microstructure of biogenic aragonite as well as calcite are distinct from those of their nonbiogenic counterparts. In the aragonitic shell of Acanthocardia tuberculata, anisotropic lattice distortions of 0.1% along the c-axis were detected.7 Furthermore, mild heat treatments led to full relaxation of the lattice distortions. These phenomena provided evidence that intracrystalline macromolecules induce distortions and that these are relaxed by their denaturation via heat treatment.7,8 By application of a focused ion beam and annular dark-field scanning transmission electron microscopy (ADF-STEM) coupled with 3D-reconstruction, Li and co-workers resolved the spatial distribution of organic matter in single crystals of synthetic calcite9 and in calcitic prisms derived from the mollusk Atrina rigida.10 The organic molecules were found to occur either as a network9 or as approximately 10 nm intracrystalline aggregates with a more circular shape along the c-axis.10 ADF-STEM coupled with 3D-reconstruction also beautifully revealed the intracrystalline organic phase within the Received: February 7, 2014 Revised: June 25, 2014

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nacreous layer in the green mussel Perna canaliculus.11 Also, atom-probe tomography turned out to be a powerful tool to visualize organic−inorganic interfaces and to characterize their local chemical environment at high resolution.12 Additional information about the binding properties of macromolecules to the crystalline phase was provided by X-ray absorption near-edge structure spectroscopy and X-ray photoelectron emission microscopy. Using the Asp2 peptide as an example, Metzler et al. demonstrated that interaction of protein with the crystal phase led to short-range disorder in the crystal structure.13 Moreover, the influence of shell extracts on mineral formation has been investigated in several studies.2−4,14−16 An acid-soluble extract of Pinctada margaritifera was recently shown to build hierarchical and nanostructured CaCO3 crystals when added to a supersaturated solution of calcium carbonate.15 Fabrication of artificial systems could be facilitated, however, by reducing their complexity through a better understanding of the protein function. This principle was adopted in quite a few studies, in which the authors used peptide-based systems,17 recombinant proteins such as Pmargpearlin,18 or the intracrystalline protein Caspartin19,20 as well as an acidic matrix protein from Pinctada f ucata.21 The last of these was identified as a key regulator of nacre formation in the Japanese pearl oyster Pinctada f ucata.21 Tuning protein properties while simultaneously conserving protein function is a highly desirable objective in the attempt to mimic natural mineral growth strategies. One fast-forward strategy is to choose a biotechnological pathway, in which protein function can be optimized with regard to specific requirements. This route offers a convenient possibility to introduce biomarkers such as fluorescent proteins or dyes for tracking the organic compound in a composite material. Even the intrinsic autofluorescence of organic compounds can help to map their distribution, as demonstrated for the calcitic shell of brachiopods.22 In general, fluorescence microscopy as well as confocal laser scanning microscopy (CLSM) are powerful methods to visualize the distribution of organic compounds in the time-course of interaction with inorganic materials such as crystals.23,24 While the green fluorescent protein (GFP) is famous for its fluorescence properties, it was recently demonstrated that it also increases the solubility of the otherwise insoluble recombinant biomineralization protein perlucin, which is the main focus of the study presented here.25 Native perlucin was extracted and purified some 15 years ago from the nacre of the small gastropod mollusk Haliotis laevigata.26 Biochemical analyses revealed that the native 17-kDa protein is glycosylated and contains a calcium-dependent C-type lectin domain.27 Further studies identified perlucin (155 amino acids) as the Nterminal part of a 240-amino-acid inmaturated protein precursor.28,29 So far, heterologous expression and purification of perlucin variants were achieved either by means of denaturation/renaturation strategies30 or by recombinant fusion to soluble protein tags such as GFP25 or the maltosebinding domain.31 Crystallization studies in vitro indicate that perlucin is incorporated into calcium carbonate and promotes calcite formation.32 A systematic approach performed by Borukhin et al. revealed that not only proteins can be incorporated into the calcite crystal lattice but also single amino acids. This type of inorganic inclusions was shown to induce anisotropic lattice distortions of up to 0.2% along the c-axis of the calcite host when grown in the presence of aspartic acid and cysteine.33 The incorporation

of cysteine was surprising, as up to now, discussions of acidic amino acids have emphasized their major role in the context in directing crystal growth.34,35 It was proposed that the formation of a calcium-thiol bond was responsible for the surprising effect of cysteine incorporation.33 There is a constant desire to develop new materials with tunable functional properties. Investigation of how organic molecules interact with inorganic matter does not only provide fundamental knowledge on crystal formation. It also opens much broader perspectives for the application of bioinspired concepts. These strategies might lead to the development of new materials with tunable functional properties. A recent experimental approach toward band gap engineering demonstrated that amino acids can become incorporated into zinc oxide crystals, inducing lattice distortions accompanied by an increase in the band gap of the semiconductor hosts.36 In the present study, we describe the effect of the biotechnologically produced His-tagged GFP-perlucin fusion protein on the crystal structure and microstructure of synthetic calcite. Using high-resolution XRD, we demonstrate that the recombinant protein induces anisotropic lattice distortions in synthetic calcium carbonate. Furthermore, His-tagged GFPperlucin was incorporated into the synthetic composite crystals to a considerably higher extent than pure His-tagged GFP. We conclude that recombinatly synthesized biomineralizationfusion proteins serve as an excellent model system for the study of protein−mineral interaction.

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EXPERIMENTAL SECTION

Protein Expression and Purification. Recombinant His-tagged GFP-perlucin was expressed and purified as described.25 In brief, Histagged GFP-perlucin expression was based on the pQE expression systems (Qiagen, Hilden, Germany) using the perlucin sequence derived from the UniProt Knowledgebase (UniProtKB), accession no. P82596. Control experiments were carried out using His-tagged GFP. The GFP sequence was encoded by the removal of the C-terminal chitin-binding domain (CBD) sequence in the pQE31-GFP-CBP vector.37 Bacteria transformed with His-tagged GFP and His-tagged GFPperlucin-encoding sequences were allowed to grow to a final optical density of approximately 2.8 and 2.3, respectively. Cells were harvested, and proteins were affinity purified using a Ni-NTA based column (Ni2+-NTA Agarose, Qiagen, Hilden, Germany) according to the manufacturer’s guidelines (QiaExpressionist). Purity of the onestep protein eluate was assessed by standard sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS−PAGE) under reducing conditions. Protein species were visualized with Coomassie blue and silver staining.38 Protein concentration was calculated photometrically by means of the Bradford assay.39 CaCO3 Precipitation Assays. Synthetic calcium carbonate biocomposites were grown in a bulk assay at room temperature, as previously described.25,40 Immediately prior to use, proteins were dialyzed against 10 mM Tris buffer at pH 8.7, (4,000−6000 Da, MWCO, Roth, Karlsruhe, Germany). All solutions were prepared in deionized water (Milli-Q, Millipore, Schwalbach, Germany) and 0.22 μm sterile-filtered. Unless otherwise stated, all chemicals were of ACS grade and were obtained from Sigma-Aldrich (Munich, Germany) or Roth (Karlsruhe, Germany). The bulk assay was performed as follows: A solution containing 6 mL of 20 mM CaCl2 and 3 mM Tris at pH 8.7 was prepared, either without protein or in the presence of 5 μg, 25 μg, or 100 μg of Histagged GFP-perlucin or 5 μg, 25 μg, or 100 μg of His-tagged GFP. Under constant stirring, 6 mL of 20 mM NaHCO3 and 3 mM Tris at pH 8.7 were added within 10 s, leading to a final volume of 12 mL and final protein concentrations of 0.4 μg/mL (5 μg), 2 μg/mL (25 μg), and 8 μg/mL (100 μg). Only freshly prepared protein solutions were B

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Figure 1. Scanning electron micrographs of synthetic calcium carbonate. Crystals were grown without any additives (A,B) or with 25 μg (C,D) and 100 μg (E,F) of His-tagged GFP as well as in the presence of His-tagged GFP-perlucin, 25 μg (G−J) or 100 μg (K,L). Note that a needle-like substructure was observed only when His-tagged GFP-perlucin was added to the reaction setup (I−L). used. Calcium carbonate crystals formed in solution were harvested after 2 days and kept in solution. The mixing procedure was carried out 10 times for each protein and concentration. High-Resolution X-ray Powder Diffraction. High-resolution XRD analysis was carried out on the ID31 beamline of the European Synchrotron Radiation Facility (ESRF), Grenoble, France. Powder samples were investigated at a wavelength of 0.399848 Å (±0.000004). All samples were measured in a borosilicate 1 mm capillary and saturated in the crystallization solution (wet state). Crystal lattice parameters were assessed by the Rietveld refinement method with GSAS software and the EXPGUI interface.41,42 Microstructure (crystallite size and microstrain fluctuations) was calculated as described previously.8 The errors were calculated assuming that the Lorenzian and Gaussian full-width at half-maximum (fwhm) are independent of the peak position.43 The errors in the strain values were set as the maximal deviations from the calculated values according to the GSAS output of lattice parameters and their errors. Confocal Laser Scanning Microscopy. CLSM was used to localize His-tagged GFP-fusion proteins within calcium carbonate precipitates. The instrument we used was a LSM 510 META (Zeiss, Oberkochen, Germany) coupled with an Axio Imager Z1 microscope equipped with a Plan-Apochromat 63×/1.4 oil DIC objective. Samples were mounted in deionized water on glass slides. For GFP excitation, we used a multiline argon ion laser (488 nm), and emission signals between 500 and 550 nm were recorded with a BP filter. IMARIS 7.6.4 software was used for image processing, and identical settings were applied to all images. Bleach experiments were conducted in 10% H2O2 for 20 and 60 min. Scanning Electron Microscopy. A high-resolution scanning electron microscope (HR-SEM, ULTRA Plus, Zeiss, Oberkochen, Germany) was used for characterization of the crystal shape and morphology. Dried samples were mounted on carbon tape prior to carbon coating (Quorum Q150T ES, East Grinstead, UK). Images were obtained in high vacuum mode at 4 kV. Protein Modeling. The 3D structure of the asialoglycoprotein receptor (1DV8) was retrieved from the PDB protein data bank (http://www.rcsb.org/pdb/explore.do?structureId=1dv8). Structural models of perlucin analogues were composed using BALLView

Software 1.4 (http://www.ballview.org/), assuming that the conserved core regions of perlucins on the primary structure level and 1DV8 also represent similarly conserved 3D structures. Protein sequences for various perlucins were retrieved by using BLAST search tools from the Genome Portal of the DOE Joint Genome Institute (http://www.jgi. doe.gov/), Swiss-Prot, and GenBank databases with access via the NCBI (http://www.ncbi.nlm.nih.gov/) server. Sequence alignments were generated by CLUSTALW2 of the EMBL-EBI (http://www.ebi. ac.uk/Tools/clustalw2/) server.

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RESULTS AND DISCUSSION Protein Expression, Purification, and Calcium Carbonate Precipitation Assay. Recombinant His-tagged GFP and GFP-perlucin were expressed in E. coli and purified (see Figure S1, Supporting Information) for investigation of their influence on the atomic structure and microstructure of synthetic calcium carbonate. Protein expression and purification have been previously described in detail.25 A one-step eluate of His-tagged GFP or His-tagged GFPperlucin (each 5 μg, 25 μg, and 100 μg) was poured into 20 mM CaCl2 solution before an equal amount of NaHCO3 solution was added. Scanning electron microscopy revealed calcium carbonate crystals of various shape but all of them consisting of calcite (see Figures S2−S3, Supporting Information). Precipitates were obtained in the absence of protein (control, Figure 1A,B) or in the presence of His-tagged GFP (GFP, Figure 1C,D). The shape of the latter ones was comparatively more elongated. The calcite cleavage plane (104) is indicated by arrows in Figure 1B and D. The GFP-derived outer crystal shapes turned out to be more irregular at higher protein concentrations (8 μg/mL (100 μg)) as can be seen in Figure 1E,F. In the presence of His-tagged GFP-perlucin (Figure 1G,H), we observed flower-shaped crystals at 2 μg/mL (25 μg). In addition, a peculiar needlelike structure was observed at a protein concentration of 2 μg/ C

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Figure 2. Confocal laser scanning micrographs of calcium carbonate precipitates. 3D confocal image stacks in the z-direction of crystals grown in the presence of either 25 μg of His-tagged GFP (A−C) or 25 μg of His-tagged GFP-perlucin (E−G). Bright-field images of identical particles are shown in D and H, respectively. Treatment with H2O2 for 20 min led to a greater reduction of fluorescence signals in His-tagged GFP crystals (C) than in precipitates containing His-tagged GFP-perlucin (G, arrows). Overlay of fluorescence signals (calculated volume, green) derived from individual optical focal planes in the z-direction and the calculated particle volume (A and E, blue).

mL (25 μg) at the top of the crystal, an area that was previously attached to the glass surface of the reaction vessel (Figure 1I,J for lager magnification). At the higher protein concentration of 8 μg/mL (100 μg), the needle-like structure was present on the entire surface of the crystal (Figure 1K,L). Different outer shapes of the calcium carbonate precipitates indicate that proteins influence crystal growth and that they probably interact in distinct ways with the mineral phase. Confocal Laser Scanning Fluorescence Analysis: Localization of His-tagged GFP-Fusion Proteins. We used CLSM to localize His-tagged GFP and His-tagged GFPperlucin within the crystalline structure of precipitates grown in the presence of 25 μg of protein. What was clearly seen at first sight in Figure 2, without any further quantification, is that His-tagged GFP forms an outer envelope around the composite crystal (Figure 2B), whereas His-tagged GFP-perlucin appears intimately linked to the mineral phase since fluorescence signals more or less represent the texture of the mineral (Figure 2F). This, in fact, encouraged the bleach treatment in order to demonstrate that the outer envelope formed by His-tagged GFP can indeed be removed (Figure 2C). The fluorescent crystal morphology in the case of His-tagged GFP-perlucin exhibited less reduction in fluorescence (Figure 2G). Moreover, the question arose as to whether an elongated bleach treatment is suitable to learn more about the internal relationship between His-tagged GFP or Histagged GFP-perlucin and the mineral phase. Note that the exact ratio between the organic and inorganic phases may depend on a large number of parameters over which it should be rather difficult to gain control. However, a more intense bleach treatment for 60 min was demonstrated to be suitable to achieve significant differences between internal localization of fluorescence signals for the different types of recombinant proteins (Figure 3). Signals derived from precipitates grown in the presence of 25 μg of His-tagged GFP (Figure 3A and C for the bright-field image) are globular in shape and are oriented randomly. In contrast, crystals gown in the presence of His-tagged GFP-perlucin show

Figure 3. Confocal laser scanning micrographs of bleach-treated particles (H2O2 for 60 min). Fluorescence distribution (A and B, green) from one optical plane is shown within the crystalline structure (calculated particle volume is shown in blue). The precipitates were grown in the presence of either 25 μg of His-tagged GFP (A) or 25 μg of His-tagged GFP-perlucin (B). Bright-field images of identical particles are presented in C and D.

a radial distribution of fluorescence signals within the crystal (Figure 3 B and D for the bright-field image). His-tagged GFP-perlucin was found to be less sensitive than His-tagged GFP to the bleach treatment, as shown by the small difference in fluorescence before and after the treatment. We suggest that intracrystalline proteins are more resistant to the bleach treatment than intercrystalline proteins. It is likely that proteins within the crystal lattice structure are protected from the bleach solution and are therefore more preserved from degradation. To find out how His-tagged GFP and His-tagged GFPperlucin interact with the crystalline phase and to further verify that the latter indeed becomes incorporated into the crystalline D

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Figure 5). Here, the strain was calculated as Δa/a = 1.6 × 10−4 (0.02%) and Δc/c = 8.9 × 10−4 (0.09%). After heat treatment,

host while the former does not, we next performed synchrotron high-resolution powder XRD. High-Resolution X-ray Powder Diffraction Analyses. Using XRD analysis, we were able to determine that the crystalline phase of calcium carbonate precipitates was calcite in all cases (see Figure S2 and S3, Supporting Information, for XRD spectra). Figure 4 shows the (104) diffraction peak for

Figure 5. Strain calculation in synthetic calcium carbonate. Calculated strain Δa/a, Δb/b (dark gray), and Δc/c (light gray) for calcium carbonate crystals grown in the presence of recombinant synthesized His-tagged GFP and His-tagged GFP-fusion proteins at a concentration of 0.4 μg/mL (5 μg), 2 μg/mL (25 μg), and 8 μg/mL (100 μg). The asterisk indicates the sample after heat treatment.

Figure 4. (104) XRD peak of crystals of synthetic calcite. Crystals were synthesized without any additive (control, red) or in the presence of 100 μg of His-tagged GFP (green) or 100 μg of His-tagged GFPperlucin before heat treatment (pink) and after it (blue). A shift to lower angles is seen exclusively for precipitates grown in the presence of His-tagged GFP-perlucin (pink). Both peak broadening and a shift back to the peak position of the control sample are observed after heat annealing (blue).

the strain decreased to a value of Δc/c = 2.3 × 10−4 and Δa/a = −1.5 × 10−4. In the presence of 100 μg of His-tagged GFP, the calculated strain was much less pronounced in both a and c directions with Δa/a = 2 × 10−5 and Δc/c = 1 × 10−4, respectively. The corresponding lattice parameters were a = 4.99074(6) Å and c = 17.06609(7) Å. Microstructure: Crystallite Size and Microstrain Fluctuations. To determine whether the proteins bind preferentially to specific crystallographic planes, we extracted the contribution of crystallite (grain) size and the microstrain fluctuations for the (012), (104), and (006) reflections of the X-ray pattern, as described previously for biogenic calcite.8 As compared to the control calcite (770−870 nm), at low protein concentrations of 0.4 μg/mL (5 μg) the grain size was reduced in His-tagged GFP to 630−740 nm. When 5 μg of His-tagged GFP-perlucin was present during synthesis, the grain size was reduced only for the (006) and the (012) planes, to 670 and 680 nm, respectively (see Figure 6). At the higher protein concentrations of 25 μg and 100 μg, the grain size was increased in His-tagged GFP samples, reaching values of 920− 1000 nm, whereas in His-tagged GFP-perlucin the grain size was strongly reduced to less than 400 nm for the highest concentration of 8 μg/mL (100 μg), most prominent for the (006) plane. In addition, the grain size belonging to the (012) and (104) planes were reduced to 550 and 490 nm, respectively. After annealing, the grain size in the 100 μg His-tagged GFPperlucin/crystals was reduced to 100 nm in the case of the (006) plane and less than 200 nm for the (012) and (104) planes (Figure 6). We further calculated the microstrain fluctuations for each individual plane (see Figure 7). Our results showed that the microstrain fluctuations increase with reduction in grain size. Owing to the presence of His-tagged GFP-perlucin, microstrain fluctuations were increased from 1.48−3.25 × 10−4 to 3.4−5.9 × 10−4 when 25 μg or 100 μg were present during synthesis for individual planes. A dramatic increase to values of 1.19−1.68 × 10−3 was observed after annealing. In the presence of His-

precipitates grown in the presence of 100 μg of His-tagged GFP (green) or 100 μg of His-tagged GFP-perlucin (before heat treatment (pink) and after it (blue)), or without any additive (control, red). A first and clear observation is a shift of the peak to a lower Bragg angle (higher d-spacing) exclusively in the case of Histagged GFP-perlucin. In addition, after heat treatment the diffraction peak is shifted back to a higher Bragg angle identical to the control position as well as to the His-tagged GFP sample position. Pronounced broadening of the peak is also visible after the heat treatment (blue graph). The presence of intracrystalline organic molecule species was previously shown to cause shifts in the XRD pattern (lattice distortions) as a result of their incorporation into the inorganic crystal host.7,8,44 These alterations in the unit cell could be almost completely reversed by application of heat treatment that was accompanied by diffraction peak broadening. To understand how these recombinant proteins are incorporated into the calcite crystal lattice and whether they interact with specific crystal planes, we calculated the lattice distortions for each crystallographic axis on the basis of Rietveld refinement. Determination of Crystallographic Parameters of Synthetic Biocomposites: Strain Calculation. In the absence of any additive, the unit cell parameters of synthetic calcium carbonate were a = b = 4.99064(8) Å and c = 17.06429(9) Å. In the presence of either His-tagged GFP or His-tagged GFP-perlucin, concentration-dependent lattice distortions, most prominent along the c-direction, were observed, with the highest distortions at His-tagged GFPperlucin concentration of 8 μg/mL (100 μg), yielding lattice parameters of a = 4.99146(8) Å and c = 17.07957(8) Å (see E

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protein receptor as well as perlucin belongs to the superfamily of C-type (calcium-dependent) lectins. Hereby, perlucin from Haliotis laevigata and the asialoglycoprotein receptor exhibit a primary sequence similarity of 34% (Figure S4, Supporting Information). Using the BALLView software package, models of the 3D structure of different perlucin analogues based on sequence alignments in comparison to the asialoglycoprotein receptor were created (Figure 8). Assuming that the core protein exhibits almost identical conformation, there are two extended loop regions, which appear to be characteristic for the perlucin proteins (see Figure 8B,C for Haliotis laevigata and Figure S5−S7, Supporting Information, for the species Haliotis diversicolor, Haliotis discus discus, and Lottia gigantea). The BALLView 3D alignments show that loop regions could fold into a more or less orthogonal arrangement. Although it remains unclear at this stage whether or not these peculiar structures play a pivotal role in mineral interaction, the fact that charged amino acids are integral parts of these loop structures suggests that this is likely (Figure 8B, amino acids in ball-andstick view; acidic residues are shown in red and basic residues in blue). The BALLView software was further used to estimate some distances between selected amino acids, in or close to the perlucin-specific loop region. Distances of approximately 10 Å, as found in several cases for charged and polar amino acid residues, would be good candidates for potential interactions with specific crystal axes such as the a- and b-axes in calcite (each 4.99 Å) or the a-axis in aragonite (4.95 Å). Note that the values of each unit cell have to be doubled in this case. In addition, observed distances of 5.6−5.7 Å may indicate a potential interaction of the protein with the c-axis of aragonite (5.74 Å). Approximate distances of the evaluated amino acids from the loop regions of perlucin analogues are summarized in Table S1 (Supporting Information). One has to take into account that these values only represent rough estimates from a static model. Significantly more accurate molecular dynamic simulations have so far not been performed.

Figure 6. Calculation of grain size for synthetic calcium carbonate. Precipitates were grown in the presence of 5 μg, 25 μg, and 100 μg of His-tagged GFP and His-tagged GFP-perlucin. Values are calculated for the (006), (012), and (104) planes derived from high-resolution XRD spectra. The asterisk indicates the sample after heat treatment.

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Figure 7. Microstrain fluctuations calculated for synthetic calcium carbonate. Precipitates were grown in the presence of 5 μg, 25 μg, and 100 μg of His-tagged GFP and His-tagged GFP-perlucin. Values are calculated for the (006), (012), and (104) reflections. The asterisk indicates the sample after heat treatment. Note that the longer error bar for the heated sample (asterisk) for the (006) plane is caused by (a) a lower peak intensity (see Figure S3D, Supporting Information) and (b) by the vertical scattering of data points.

CONCLUSIONS The results of this study demonstrated, for the first time, that biotechnologically produced His-tagged GFP-perlucin becomes incorporated into the lattice of calcite and induces lattice distortions similar to those shown to exist in its biogenic counterpart. Although recombinant proteins differ from native proteins extracted from organisms with regard to posttranslational and sequence modifications (glycosylation, fusion proteins, and His-tag), we found that His-tagged GFP-perlucin interacts with the calcium carbonate phase in a manner comparable to that of native biomineralization proteins.7,8,44,46 In fact, in our study the highest strain values were detected along the c-axis of the crystal host. One would assume that macromolecules preferentially interact with the positively charged (001) basal plane due to the presence of acidic molecules. Indeed, these results are in good agreement with previous results from small-angle-X-ray scattering (SAXS).47 The new results for recombinant GFP-perlucin, which was shown here to modify the crystal lattice of calcite, contribute to a deeper understanding of biomineralization-specific protein function. Although it was previously found that His-tagged GFP delays calcium carbonate formation,5 the GFP domain (28.9 kDa) does not seem to modify a calcite mineral in terms of lattice distortions. At least few additional amino acids such as the N-terminus of perlucin are required to induce lattice

tagged GFP, we obtained values in the range of 1.31−3.25 × 10−4, which are in the same order of magnitude as in the case of the control sample. Our results indicated that His-tagged GFP-perlucin and Histagged GFP interact in a distinct manner with the mineral phase. Lattice distortions were anisotropic and dominant along the c-direction when His-tagged GFP-perlucin was present during crystal synthesis. In addition, grain size was reduced, and microstrain fluctuations were increased. After annealing, the grain size was further reduced owing to the formation of new interfaces, which appeared when the His-tagged GFP-perlucin was damaged by heat treatment. Model of Perlucin 3D Structure. To answer the question as to why His-tagged GFP-perlucin is incorporated into crystal lattice structure rather than His-tagged GFP, we performed modeling studies of perlucin based on the resolved 3D structure of the asialoglycoprotein receptor.45 The asialoglycoF

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Figure 8. (A) 3D structure of the asialoglycoprotein receptor45 (chain A, PDB: 1DV8, A, shown in magenta) merged with the model of perlucins structure from Haliotis diversicolor (white), Haliotis discus discus (cyan), Haliotis laevigata (green), and Lottia gigantea (orange). Specific loop regions were identified exclusively in perlucin analogues (arrows) but not in the asialoglycoprotein receptor. (B) These regions contain negatively (red) and positively (blue) charged amino acids and are represented in ball and stick view in the 3D structure of perlucin from Haliotis laevigata (green). The asialoglycoprotein receptor is drawn in magenta. (C) Distances between highlighted acidic amino acids were calculated based on the carbon atom of the carboxyl group of Glu 58 and Asp 108 (both atoms are highlighted in yellow). The distance between them was determined to be 10.832385 Å. All Figures were drawn using the program BALLView. Comparative data of perlucin analogues are provided as Supporting Information (Figures S4− S7 and Table S1).

distortions, as unequivocally shown here. Further, the absence of glycosylation in the recombinant perlucin may indicate that the sugar modification is related to additional functions such as modulating protein solubility. Previous findings25−27,30,32 as well as this study suggest that the natural function of perlucin is less likely related to polymorph control in the calcite/aragonite system, taking into account that native perlucin was originally extracted from aragonite shells. Some molecular features of perlucin, obtained from a correlative 3D model of perlucin analogues, based on the structure of a highly conserved C-type lectin core domain, identified variable loop regions. These regions seem to form distinct features of perlucin derivatives from various species. Whether or not these peculiar loops are in fact important for species-specific protein function in terms of shell structure and/ or intracellular signaling remains so far an open question. The presence of charged amino acids in surface-exposed extended loop structures of the perlucins provide at least a very first hint for a distinct interrelationship with the observed lattice distortions in GFP-perlucin-induced calcite composites. Although native perlucin could differently interact with the fine-tuning of crystal morphologies and perhaps interfere with

intracellular signaling in living systems in a completely different manner, this work on recombinant perlucin and its direct modulation of the crystallographic properties of calcite represents a first step toward biotechnological GFP-crystal engineering based on a rational design approach. Notably, recombinant perlucin without the GFP-tag is insoluble under native conditions. It would nevertheless be worthwhile to investigate whether protein modifications can be used to tune protein properties and protein−mineral interaction, similar to the example of calcium carbonate/ silicatein-α biocomposites with enhanced bending strength and light-wave guiding properties,48 in terms of targeting a broader range of mechanical and optical properties. Taking all of these findings into account, we believe that recombinant biomineralization-fusion proteins have the potential to open up promising strategies for directing protein− mineral interaction in specific ways, allowing the design of new materials with extraordinary properties. G

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ASSOCIATED CONTENT

S Supporting Information *

Silver stained SDS−PAGE of purified His-tagged GFP and Histagged GFP-perlucin as well as high-resolution powder XRD pattern of all calcite samples, and modeling results of the 3D structure of perlucin proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement [ number 336077]. We also thank the Minerva foundation for financial support. We greatly appreciate the help of Marc Thobae and Frederik Schweiger in data acquisition, as well as the support of Dr. Nitsan Dahan from the LS&E Infrastructure Unit of the Technion-Israel Institute of Technology. We thank Professor Eduard Arzt from the INMLeibniz Institute for New Materials for his support.

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ABBREVIATIONS CLSM, confocal laser scanning microscope; GFP, green fluorescent protein; XRD, X-ray diffraction; 3D, 3-dimensional protein simulation

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REFERENCES

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dx.doi.org/10.1021/cm500450s | Chem. Mater. XXXX, XXX, XXX−XXX