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Accelerated nucleation of hydroxyapatite using an engineered hydrophobin fusion protein Melanie Melcher, Sandra J. Facey, Thorsten M. Henkes, Thomas Subkowski, and Bernhard Hauer Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00135 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 27, 2016

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Accelerated nucleation of hydroxyapatite using an engineered hydrophobin fusion protein

Melanie Melcher1, Sandra J. Facey1, Thorsten M. Henkes1, Thomas Subkowski2 and Bernhard Hauer1*

1

Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart,

Germany 2

Fine Chemicals Research, BASF SE, 67056 Ludwigshafen, Germany

* Corresponding author: Bernhard Hauer, Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany, Phone: 0049-711-685-63193; Fax: 0049-711-685-64569; E-mail: [email protected]

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Abstract Calcium phosphate mineralization is of particular interest in dental repair. A biomimetic approach using proteins or peptides is a highly promising way to reconstruct eroded teeth. In this study, the screening of several proteins is described for their binding and nucleating activities towards hydroxyapatite. Out of 27 tested candidates only two hydrophobin fusion proteins showed binding abilities to hydroxyapatite in a mouthwash formulation and an increased nucleation in artificial saliva. Using a semi rational approach, one of the two candidates (DEWA_5), a fusion protein consisting of a truncated section of the Bacillus subtilis synthase YaaD, the Aspergillus nidulans hydrophobin DEWA and the rationally designed peptide P11-4 described in the literature could be further engineered towards a faster mineral formation. The variants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance the nucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase in the binding towards hydroxyapatite could be achieved. Especially with the variant DEWA_5a the protein engineering of the rationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineered peptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis.

Keywords:

Biomimetic

material, Biomineralization, Hydrophobin, Hydroxyapatite,

Calcium

phosphate, XRD

Introduction The mammalian tooth is composed of several tissues. One of them is enamel, the hardest material in the vertebrate body.1 Enamel contains a high amount of hydroxyapatite, approximately 95%. In enamel, the hydroxyapatite nanocrystals are highly organized and elongated in the direction of the caxis.2 The extreme hardness and stability of the dental enamel is attributed to this special structure. 2 ACS Paragon Plus Environment

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Though, because of the high content of inorganic material and the absence of organic compounds, like polymers and proteins, there is no in vivo mechanism to reconstruct the dental enamel once destroyed. Required is an approach to form enamel like material in a synthetic manner. In the past, many research groups have focused on the in vitro synthesis of enamel.1,3–12 Protein or peptide mediated biomineralization has been found to be a promising method to reconstruct enamel because it can occur under physiological conditions to provide a biocompatible material. Detailed studies of extracellular matrix proteins during the development of enamel or dentin have led to a deeper understanding of the biomineralization process of teeth.8,9,13–15 The major proteins amelogenin and dentin phosphoprotein have been found to play an important role in modulating the mineralization of organized calcium phosphate crystals. Amelogenin is the major protein component of the enamel extracellular matrix during enamel formation. The self-assembly of amelogenin into nanospheres is believed to play a key role in controlling the oriented and elongated growth of the hydroxyapatite crystals.16,17 In the case of dentinogenesis, nucleation and growth of hydroxyapatite is mainly attributed to non-collagenous proteins that are rich in acidic amino acids.18 One of these noncollagenous proteins of the dentin extracellular matrix is the dentin phosphoprotein (DPP). It contains an unusually high amount of phosphorylated serine residues and aspartic acids with approximately 90%.19 In the presence of Ca2+ ions DPP self-assembles into a β-sheet-like conformation, serving as a high negatively charged surface. This surface may not only interact with the growing hydroxyapatite crystal, but also be involved in the first crystal deposition during dentinogenesis.20–22 Also in vitro experiments have demonstrated the mineralizing effect of immobilized DPP.23,24 These abilities may be attributed to the predominate motif D-S-S in DPP.25 Yarbrough et al.9 have further demonstrated the binding abilities of peptides containing a different number of the predominant motif D-S-S (DSS)n without phosphorylation to hydroxyapatite and mineralized tissues. They also showed the mineralizing effect of (DSS)8 after immobilizing onto polystyrene beads, whereas in free solution these peptides failed to enhance mineralization. Also the


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unphosphorylated triple repeat (DSS)3 promotes apatite deposition on demineralized enamel after binding of the peptide to eroded teeth.11 Based on the knowledge that these proteins present favorable sites for nucleation in ordered and rigid conformation, there have been approaches to mimic the extracellular matrix (ECM) of such systems by the rational design of peptides.12,26–28 One example is the rationally designed selfassembling peptide P11-4.12 This anionic peptide can self-assemble into β-sheet lattices that further assemble into a fibrillar network. These networks serve as a scaffold for crystal deposition like ECM does in nature. Furthermore, Kirkham et al.12 demonstrated a remineralization effect of this peptide in dental lesions. For clinical applications or oral care products, different approaches have been developed to treat eroded enamel or caries lesions. Fluoride was identified to be an effective remineralizing agent.29 This was due to the substitution of hydroxyl groups by fluoride ions in which fluorapatite (Ca10(PO4)6F2) is formed. This shows a higher stability and is insoluble in a broader pH range.30–32 Nanohydroxyapatite shows a crystal structure similar to that of dental apatite.33,34 Many efforts demonstrated that nano-sized hydroxyapatite shows a positive effect on the remineralization of the eroded enamel and caries lesions.33,35 Furthermore, phosphopeptides derived from casein were applied in combination with amorphous calcium phosphate (CPP-ACP) in oral care products.36 In clinical studies, the remineralizing potential of CCP-ACP was demonstrated.32,37 But these approaches also inherit problems. There is still a requirement to improve the delivery of fluoride in optimal amounts to overcome high risk caries in many individuals.38 An excessive uptake may result in enamel fluorosis, especially in children’s teeth.39–41 A biomimetic approach would lead to a direct reconstruction of the decayed enamel structure. This study seeks to identify and characterize proteins that are able to remineralize hydroxyapatite in vitro. Therefore, a panel of various fusion proteins was characterized in regard of their binding and mineralization abilities. The panel resulted from 4 different proteins, which were used in their native form, as cleavage products or as fusion proteins in combination with literature described peptides. 4 ACS Paragon Plus Environment

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Amelogenin and statherin were chosen because of their described binding activities to hydroxyapatite,42,43 whereas anti-freeze proteins and the Aspergillus nidulans hydrophobin DEWA (formerly known as CAN4)44 were chosen because of their well-known surface active abilities.45-47 DEWA was also previously reported to be expressible in E. coli when being expressed as a fusion protein with the Bacillus subtilis synthase YaaD, which allows it’s usage in industrial applications.48 All tested hydrophobin DEWA-constructs contained an amino-terminal fusion to the Bacillus subtilis synthase YaaD or a truncated version consisting of 40 amino acids of YaaD as described as H*protein A or B by Wohlleben and co-workers, respectively.48 In this study, we investigated the ability of the different fusion proteins and peptides to bind to hydroxyapatite in HBS-T buffer and in a mouthwash formulation. Furthermore, nucleation activities of these proteins were analyzed and characterized using X-ray diffraction and transmission electron microscopy. For a more detailed determination of the crucial amino acids needed for nucleation, our best candidate, a fusion construct of the peptide P11-4 and the fusion protein consisting of hydrophobin DEWA and the truncated version of YaaD, was further investigated by site-directed mutagenesis. Based on the sequence of P11-4 the nucleation activity was enhanced by different mutagenesis strategies.

Experimental Section Materials The fusion proteins consisted of nine amelogenin constructs, nine antifreeze constructs, five hydrophobin constructs and four statherin variants. Peptides extracted out of commercially available MI tooth mousseTM ,49 (DSS)6 9 and peptide #3-1 (LIKHILHRL),9 which was described as a non-binder towards hydroxyapatite, were used as controls. All 30 proteins and peptides were obtained purified or as lyophilized powder, respectively, from the BASF SE (Ludwigshafen, Germany) (Table 1).

Bacterial strains and culture conditions 5 ACS Paragon Plus Environment

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E. coli XL1-blue was used for routine subcloning and plasmid propagation. The E. coli strain M15 [pREP4]50 was used for expression of the recombinant proteins in the vector pQE60 (Qiagen, Hilden, Germany). Media preparation and bacterial manipulations were performed according to standard methods.51 Where appropriate, ampicillin (100 µg/mL, final concentration), and kanamycin (25 µg/mL, final concentration) were added to the medium.

Construction of hydrophobin variants based on DEWA_5 The construct DEWA_5 containing the sequence encoding for 40aaYaaD-P11-4-DEWA, consisting of the first 40 amino acid residues of the synthase YaaD of Bacillus subtilis for recombinant expression in E. coli,48 the 11-mer hydroxyapatite binding peptide P11-412 and hydrophobin DEWA, was kindly provided by BASF. For the alanine scan, the codons for the amino acids of the P11-4 peptide sequence, QQRFEWEFEQQ, between amino acid positions 49 and 59 in DEWA_5 were exchanged to codons for alanine by site-directed mutagenesis using the QuikChange method (Stratagene). To identify which part of the P11-4 sequence is important for nucleation deletion variants were constructed. Based on the truncated variant ∆3 (40aaYaaD-FEWEFE-DEWA), a small focused library was created. Negatively charged glutamate residues were exchanged either to aspartate or glutamine. The aromatic amino acids (phenylalanine/tryptophan) were exchanged to hydroxylated (tyrosine/serine), positively charged (histidine/arginine) and nonpolar (valine) residues. All constructs were confirmed by DNA sequencing.

Expression and purification For expression of the targeted proteins, the plasmids were transformed by heat shock into the E. coli strain M15 [pREP4].50 A single colony was cultured overnight in 3 mL of EC3 media (15 g/L yeast extract, 15 g/L tryptone, 30 g/L glycerol, 11.5 mM K2HPO4, 37.8 mM (NH4)2SO4, 4.1 mM MgSO4 x 7H2O, 0.7 mM CaCl2 x 2H2O, 10 mL/L 5x trace element solution SL4) supplemented with 100 µg/mL ampicillin and 25 µg/mL kanamycin. Trace element solution SL4 consists of 1.5 g/L Titriplex III, 1.0 g/L 6 ACS Paragon Plus Environment

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FeSO4 x H2O, 50 mg/L ZnSO4 x 7 H2O, 15 mg/L MnCl2 x 4 H2O, 150 mg/L H2BO3, 100 mg/L CoCl2 x 6 H2O, 5 mg/L CuCl2 x 2 H2O, 10 mg/L NiCl2 x 6 H2O and 15 mg/L Na2MoO4 x 2 H2O. Overnight cultures were diluted to an OD600 of 0.5 in fresh EC3 media containing the appropriate antibiotics and cultivated at 37 °C, 180 rpm for 90 min. Expression of the recombinant proteins was induced with isopropyl-ß-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After additional incubation for 3 h at 37 °C, 180 rpm the cells were harvested by centrifugation for 20 min, 6000 rpm at 4 °C. The DEWA_5 variants were purified by renaturing the inclusion bodies. Therefore cells were disrupted by sonification, washed with TBS buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5) and centrifuged (10 min, 8000 rpm, 4 °C). The supernatant was discarded and the obtained inclusion bodies were renaturated by the addition of NaOH to a final concentration of 0.1 M and stirring for 10 min at room temperature. The pH was adjusted to 7.5 by titration with 2% H3PO4 and insoluble proteins were separated by centrifugation (20 min, 8000 rpm, 4 °C). After dialysis of the supernatant against 5 mM Tris-HCl, pH 7.5, MgCl2 was added to a final concentration of 4.2 mM and DNA was removed by a treatment with DNAse I for 1 h at room temperature. For nucleation, proteins were again dialyzed against 5 mM Tris-HCl, pH 7.5. Protein expression was analyzed by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Gels were stained with Coomassie Brilliant Blue (R 250).

Protein labeling For binding studies 5(6)-carboxyfluorescein was conjugated to the N-terminus of the proteins by using the corresponding succinimidyl ester (Invitrogen, Darmstadt, Germany). The labeling reaction was performed according to the manufacturer’s protocol (Invitrogen, Darmstadt, Germany). After conjugation, the labeled protein was purified either with a PD-10 desalting column for proteins larger than 5 kDa or a PD MidiTrap G-10 for smaller proteins (GE Healthcare, Buckinghamshire, UK). Protein


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concentration was determined by the bicinchoninic acid method using the BCA Protein Assay Kit (Thermo Scientific, Dreieich, Germany).

Binding to hydroxyapatite powder For the binding experiments N-terminal labeled proteins were used in a final concentration of 2 µM. The binding was carried out in HBS-T buffer (150 mM NaCl, 50 mM HEPES pH 7.5, 0.1% Tween 20) and a mouthwash formulation (6.0 mM NaF, 0.5 mM sodium saccharine, 16.7 mM NaH2PO4, 28.1 mM Na2HPO4, 407.2 mM glycerol, 492.8 mM propylene glycol, 150.9 mM sorbitol, 0.5% Poloxamer 407). The proteins were exposed to 10 mg hydroxyapatite powder (Fluka) for 2 h at 37 °C. After five wash steps with HBS-T buffer the bound protein was visualized by fluorescence microscopy with the appropriate filters (excitation 475/50; emission 525/50). To calculate the amount of bound protein, the fluorescence intensity of the solution was measured before (Fi) and after (Ff) the binding reaction with Fluostar Galaxy (BMG, Ortenberg, Germany). The amount of bound protein cbound was calculated according to the equation: cbound=[1-(Ff/Fi)]xc0, where c0 is the initial protein concentration. The peptide #3-1 (LIKHILHRL), which was described as a non-binding peptide to hydroxyapatite, was used as a negative control.9 All binding experiments were carried out as triplicates.

Binding to human teeth slices The binding of the fusion proteins to human teeth in the mouthwash formulation was also investigated. Adult human teeth were sagittal sectioned into slices, 100 µm in thickness, using a microtome (Institute of Mineralogy, University of Stuttgart). Prior to the use, the tooth slices were rinsed with deionized water and equilibrated for 30 min in mouthwash formulation. After incubation for 1 h in mouthwash formulation containing 2 µM of labeled protein, the tooth slices were rinsed again with deionized water and imaged by fluorescence microscopy.

Nucleation assays 8 ACS Paragon Plus Environment

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To determine the nucleating abilities of the proteins the decrease in Ca2+ concentration was measured. Nucleation was carried out in artificial saliva52 containing 8.7 mM KCl, 0.6 mM MgCl2, 1.5 mM CaCl2, 4.6 mM K2HPO4, 2.7 mM KH2PO4 and 25 µM protein in a final volume of 1 mL. As a negative control only the nucleation solution without any protein was used. All solutions were filtered (0.2 µm) prior to use to avoid uncontrolled nucleation due to small particles. Periodically, 30 µL samples were taken every hour for a total of 5 h, centrifuged 2 min, 13000 rpm and the calcium concentration of the supernatant was determined. For the calcium detection, 30 µM ocresolphthalein, 2.7 mM 8-hydroxyquinoline, 20 mM 2-amino-2-methyl-1-propanol pH 10.5 and 20 µL of the sample were mixed and the absorbance was monitored at 575 nm using a Spectramax (Molecular Devices, Biberach, Germany). All nucleation experiments were carried out at 37 °C in triplicates.

X-ray powder diffraction For characterization of the hydroxyapatite crystals, the nucleation assay was scaled up to a final volume of 50 mL. The formed precipitate after 4 h was centrifuged and rinsed five times with deionized water to remove remaining ions. The mineral was dried and characterized by X-ray powder diffraction analysis using a Bruker D8 Advance diffractometer of Cu Kα radiation at 1.54Å with a scanning rate of 0.015 °/step with 2Ѳ in the range from 24° to 67°. The experimental patterns obtained were compared with a hydroxyapatite standard (card No. 09-0432) compiled by the Joint Committee on Powder Diffraction and Standards (JCPDS).

Transmission electron microscopy (TEM) Nucleation was stopped after 4 h by centrifugation of the nucleation mixture. The mineral was rinsed five times with deionized water and resuspended in water. A 5 µL sample was applied to a formvar/carbon coated Cu 100-mesh TEM grid (Plano, Wetzlar, Germany) and air-dried. Imaging was performed using a Tecnai G2 Sphera TEM at 120 kV. 9 ACS Paragon Plus Environment

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Results Selection of hydroxyapatite-binding proteins A panel of fusion proteins containing amelogenin, statherin, antifreeze proteins or hydrophobin was screened for their binding activities to hydroxyapatite in HBS-T buffer and a mouthwash formulation. The fusion proteins were labeled with 5(6)-carboxyfluorescein and exposed to hydroxyapatite powder. The quantitative binding results are summarized in Table 2. Binding to hydroxyapatite was detected in all fusion proteins in HBS-T buffer except for the negative control peptide #3-1 (LIKHILHRL) which was described in the literature9 as a non-binding peptide for hydroxyapatite. Both positive controls, MI tooth mousseTM 49 and (DSS)6,9 showed good binding with more than 70% bound peptide. In the group of the amelogenin derivatives the amount of bound protein to hydroxyapatite was in the range of 60% except for the full length amelogenin (Amelo_1) and the fusion construct consisting of the titania mineralizing peptide R5 (SSKKSGSYSGSKGSKRRIL)53 (Amelo_9). Both constructs, Amelo_1 and Amelo_9, showed only minor binding activity in HBS-T buffer. In the case of the antifreeze fusion proteins, binding was detected in the range of 34% for the antifreeze protein 4 from Choristoneura fumiferana (AFP_1) and 91% for the antifreeze protein from Tenebrio molitor (AFP_9) with the fusion partner CherryTM tag (Delphi Genetics SA, Charleroi, Belgium). Also the hydrophobin constructs showed good binding abilities between 30% for DEWA_1, consisting of the full length Bacillus subtilis synthase YaaD and the hydrophobin DEWA from Aspergillus nidulans and 80% for the fusion construct of a truncated version of YaaD, the hydroxyapatite binding peptide CPL12 (QPYHPTIPQSVH)54 and DEWA (DEWA_4). The group of statherin fusion proteins revealed good binding to hydroxyapatite with more than 70% bound protein except Stat_2, the unphosphorylated statherin peptide, with only 16%. A completely different binding behavior for the fusion proteins was observed when the HBS-T buffer was exchanged with the mouthwash formulation. The positive control MI tooth mousseTM revealed binding to hydroxyapatite of only about 30%. In contrast, the other positive control, (DSS)6 lost its binding activity completely. Also, in the amelogenin group, no binding to hydroxyapatite was 10 ACS Paragon Plus Environment

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detected. For the antifreeze fusion proteins a similar behavior could be observed. Out of the nine proteins, only two showed moderate binding towards hydroxyapatite in the mouthwash formulation (AFP_7, AFP_8). AFP_9, the antifreeze protein from T. molitor, which had excellent binding capabilities in HBS-T buffer revealed only a minor activity (3%) in the mouthwash formulation. In the group of the statherin variants, the phosphorylated Stat_3 showed moderate binding to hydroxyapatite with about 14%. In contrast, the fusion proteins containing hydrophobin kept their binding ability. Only DEWA_1, consisting of the full length YaaD and the hydrophobin DEWA, lost its binding activity. DEWA_2, containing a truncated version of YaaD, maintained a moderate binding with about 25%. Fusing the titania mineralizing peptide R553 to DEWA_2, led to a loss in the amount of the bound protein to 2% (DEWA_3). Regarding the constructs DEWA_4 (40aaYaaD-CPL12-DEWA) and DEWA_5 (40aaYaaD-P11-4-DEWA), binding could be increased to about 36% and 30%, respectively, by inserting a hydroxyapatite binding peptide. This is in the range of the positive control MI tooth mousseTM (30%). The quantitative binding results were confirmed by fluorescence microscopy (data not shown). The proteins which were able to bind to hydroxyapatite in the mouthwash formulation were also tested for their binding ability to human tooth slices. For Stat_3, DEWA_2, DEWA_4, DEWA_5, and the positive control MI tooth mousseTM, a qualitative binding to the tooth slices could be visualized by fluorescence microscopy. Binding was mainly detected towards dentin, only minor binding was observed to enamel. The negative control, peptide #3-1, showed only a small amount of unspecific binding towards dentin and enamel (Figure 1).

Analysis of nucleation ability To determine if the different fusion constructs may enhance the formation of hydroxyapatite, the proteins were incubated in artificial saliva containing 1.5 mM Ca2+ and 7.3 mM PO43-. Mineral formation was monitored by the detection of the decrease in c(Ca2+) in the artificial saliva. The consumption of Ca2+ ions in the solution is correlated with the formation of a Ca-containing mineral 11 ACS Paragon Plus Environment

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and is therefore an indication for nucleation. Analyzing the time dependent deposition of mineral we observed three different trends (Table 2). In the presence of no protein, the mineral formation was completed after four hours, where a stable c(Ca2+) was reached. The majority of the investigated proteins exhibited the same nucleation behavior like the control without protein. The four proteins (DEWA_3, Stat_1, Stat_3 and Stat_4) showed a stabilizing effect, resulting in a deceleration in nucleation (Supporting Information, Figure S1). The phosphorylated statherin peptide Stat_3 decelerated the nucleation, whereas the unphosphorylated peptide (Stat_2) showed no effect in nucleation. Also the peptides from MI tooth mousseTM, one of the best binders in the mouthwash formulation, exhibited a strong stabilizing effect. Out of the nine antifreeze fusion proteins, four variants (AFP_1, AFP_2, AFP_4, AFP_5) showed an increase in nucleation activity but no binding activity in the mouthwash formulation. For these proteins, almost all the Ca2+ was consumed after three hours, as compared to four hours for the control without protein (Figure 2a). An even stronger effect in nucleation activity was observed with the two hydrophobin constructs DEWA_4 and DEWA_5 containing the hydroxyapatite binding peptides, CPL1254 and P11-4,12 respectively. After two hours almost no free Ca2+ could be detected in the nucleation solution (Figure 2b). The precipitate formed in the presence of the construct DEWA_4 or DEWA_5 and a control with no protein was characterized by X-ray powder diffraction. The mineral phase, including the control without protein, could be identified as crystalline hydroxyapatite. Major peaks were detected at 2Θ =25.9 ° and 2Θ = 32.9° corresponding to the (002) and the (211)/(112) plane of hydroxyapatite (Figure 3). Transmission electron microscopy of the formed mineral showed plate-like crystals that is consistent with the morphology of hydroxyapatite. The crystals were similar in size, but showed a difference in the crystal arrangement. The control sample without protein showed a spherical arrangement of the crystal, whereas the crystals formed in the presence of DEWA_4 and DEWA_5 showed more a bundle-like arrangement (Figure 4).

Investigation of the crucial amino acids involved in nucleation 12 ACS Paragon Plus Environment

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The results of the binding and the nucleation experiments showed that only two proteins, namely DEWA_4 and DEWA_5, have binding and nucleation activities in respect to hydroxyapatite (see Table 2). These two proteins share the same core structure, consisting of a truncated version of YaaD, a hydroxyapatite binding peptide and a hydrophobin DEWA at the C-terminus of the construct. They differ only in the sequence of the peptide. DEWA_4 includes the peptide sequence of CLP12 (QPYHPTIPQSVH) identified by Chung et al.54 via phage display, whereas DEWA_5 contains the sequence of P11-4 (QQRFEWEFEQQ), a rationally designed peptide to form hydrogels.12 The construct DEWA_2 (40aaYaaD-DEWA) consisting of a truncated section of the Bacillus subtilis synthase YaaD and the Aspergillus nidulans hydrophobin DEWA without an inserted peptide sequence showed good binding to hydroxyapatite in the mouthwash formulation with about 25% bound protein. Inserting the CLP12 peptide or the P11-4 peptide enhances the binding capability to hydroxyapatite. Of all the investigated proteins DEWA_4 and DEWA_5 showed the fastest decrease in calcium concentration, whereas the construct DEWA_2 had no effect on the nucleation (see Figures 2b and 5). This suggests that the hydrophobin protein, DEWA, and the truncated YaaD in DEWA_4 and DEWA_5 is responsible for the binding to hydroxyapatite, whereas the inserted peptides in the constructs are responsible for the nucleation activities. P11-4 by itself showed no effect on the nucleation (Figure 5). Beside binding capabilities, the fusion construct of the hydrophobin and the truncated YaaD seems to act as kind of a scaffold for nucleation, stabilizing the inserted peptides. The construct DEWA_5 containing the sequence of P11-4, QQRFEWEFEQQ, was chosen for further investigations. In order to determine which amino acid residues play an important role for nucleation an alanine scan was performed on the sequence of P11-4 within DEWA_5. Eight single, two double and one triple mutant were constructed (see Figure 6a). In addition, truncated versions of DEWA_5 were also constructed. Amino acid residues of the sequence of P11-4 in DEWA_5 were stepwise deleted (see Figure 6a). The expressed variants of the single alanine substitutions showed no effect on the nucleation behavior in artificial saliva. All tested single variants showed a comparable nucleation trend as 13 ACS Paragon Plus Environment

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DEWA_5 (Supporting Information, Figure S2). Also the double substitution of Q49 and Q50 with alanine had no effect on the nucleation behavior. The minor impact of the glutamine residues on the nucleation activity was further demonstrated by the deletion variants. Deletion of the two residues Q49 and Q50, resulting in variant ∆1, as well as the additional deletion of the residues Q58 and Q59 (∆2) had no effect on the nucleation ability of the construct. Also variant ∆3 (40aaYaaD-FEWEFEDEWA), containing only six of the originally eleven amino acid residues of P11-4, showed the same nucleation trend as the wild-type construct DEWA_5, containing the whole P11-4 peptide sequence (Supporting Information, Figure S3). The importance of the glutamate residues within the sequence of P11-4 for the nucleation activity of the construct DEWA_5 was clearly demonstrated by the alanine and deletion variants. Single alanine mutations at the positions E53, E55 or E57 did not influence the nucleation behavior. But the double substitution of EE53/55AA resulted in a slightly slower mineral formation. The substitution of all three glutamate residues (EEE53/55/57AAA) showed a significant deceleration in mineral deposition. The deletion variant ∆4 lacking the third glutamate residue at position 57 exhibited a slightly delayed nucleation trend as compared to the variant Δ3 or the wild type construct DEWA_5. Deletion of the second glutamate residue at position 55 (variant ∆5) led to a significantly slower nucleation (Figure 7). Since the amino acid sequence of P11-4 can be reduced to six amino acids without losing nucleation activity, the truncated variant Δ3 (40aaYaaD-FEWEFE-DEWA) was chosen to engineer the protein to a faster nucleation activity. Based on the six amino acid residues FEWEFE within Δ3, a small focused library was designed (see Figure 6b). Even if the alanine scan as well as the deletion variants demonstrated the importance of the glutamate residues within P11-4 (QQRFEWEFEQQ), glutamate residues in variant Δ3 (40aaYaaD-FEWEFE-DEWA) were exchanged into the uncharged homolog glutamine. Furthermore, glutamate was replaced by aspartate which has a shorter side chain. In order to determine the effect of aromatic and non-aromatic amino acids on the nucleation activity, the aromatic amino acids phenylalanine and tryptophan in the FEWEFE-sequence were replaced either by histidine to introduce a positively charged residue or by tyrosine to introduce a 14 ACS Paragon Plus Environment

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hydroxylated residue. In addition, the aromatic amino acids were exchanged to serine, arginine and valine. A single substitution of glutamate to glutamine (E50Q) caused a deceleration in nucleation (Figure 8). These results support that the negative charge is important for the nucleation activity. Similarly an exchange into aspartate (E50D) resulted in a slightly faster mineral formation. The replacement of all three glutamate residues in the FEWEFE-sequence of the variant Δ3 by aspartate (EEE50/52/54DDD) demonstrated more clearly the beneficial effect of aspartate on the nucleation activity (Figure 8). In the case of the aromatic amino acids phenylalanine and tryptophan a slightly higher nucleation rate was observed by introducing serine and arginine (Supporting Information, Figure S4). The replacement of F49, W51 or F53 with the aromatic homologs tyrosine and histidine did not show any effect (Supporting Information, Figure S5). These variants had a similar nucleation behavior like DEWA_5. Also the substitution to valine had no effect on the nucleation. The nonaromatic amino acid residues seem to be more beneficial for the nucleation than the aromatic residues. The final engineering step involved combining the beneficial substitutions of glutamate to aspartate and of the aromatic amino acid residues phenylalanine and tryptophan to either serine or arginine into two new constructs named DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaDRDRDRD-DEWA) (see Figure 6b). A further increase in nucleation activity in artificial saliva was observed in the presence of these two variants as compared to DEWA_5. Nucleation reached a stable plateau after 90 min with these new variants whereas with DEWA_5 nucleation was completed after 120 min (Figure 9). The analysis by X-ray powder diffraction confirmed the formed mineral as crystalline hydroxyapatite. Major peaks were observed at 2Θ =25.9 ° and 2Θ = 32.9° corresponding to the (002) and the (211)/(112) plane of hydroxyapatite (Figure 10). These variants were also tested regarding their binding abilities in HBS-T buffer and the mouthwash formulation. For the variant EEE50/52/54DDD (40aaYaaD-FDWDFD-DEWA) a slightly higher binding was observed with 76±5% in HBS-T buffer and 41±3% in the mouthwash formulation as compared to DEWA_5 with 71±6% in HBST buffer and 30±2% in the mouthwash formulation. By introducing the serine residues, the binding 15 ACS Paragon Plus Environment

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could not be further enhanced regarding the variant DEWA_5a (73±4% in HBS-T buffer, 36±4% in the mouthwash formulation). But the substitution of the aromatic amino acid residues by arginine in variant Δ3 led to a slightly higher binding of DEWA_5b with 82±3 in HBS-T buffer and 45±3% in the mouthwash formulation as compared to DEWA_5 (Figure 11).

Discussion In order to identify new potential candidates in the field of biomineralization of hydroxyapatite a panel of fusion proteins were screened for their binding and nucleating abilities. Out of the nine hydroxyapatite binding proteins in the mouthwash formulation only two fusion constructs (DEWA_4, DEWA_5) showed an increase in nucleation. That a strong binding ability does not necessarily correspond to a mineralization directing activity has been described earlier.6 It has been suggested that proteins with low affinity towards hydroxyapatite might create a local supersaturation by interacting with soluble ions and thereby reduce the nucleation barrier.6 This might be the reason for the low binding affinity of the antifreeze proteins used in this study on the one hand and the increase in nucleation on the other. In contrast, the peptides from MI tooth mousseTM and Stat_3, the phosphorylated statherin peptide, revealed good binding abilities to hydroxyapatite in the mouthwash formulation, but exhibited a strong stabilizing effect on the nucleation. The peptides in MI tooth mousseTM are derived by trypsin digestion of the milk protein casein,55 whereas Stat_3 consists of the 21 N-terminal amino acid residues of salivary statherin. Both proteins contain high amounts of charged residues like phosphoserine and aspartate residues that are known to interact strongly with calcium and phosphate ion clusters.32,56 The interaction prevents further growth of the ion clusters, resulting in stabilization57 and making milk and saliva a supersaturated solution. It is probably not surprising that the peptides from MI tooth mousseTM and Stat_3 revealed a strong stabilizing effect in our nucleation experiments. In contrast, the unphosphorylated Stat_2 did not exhibit binding abilities in the mouthwash formulation and showed no effect in the nucleation


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behavior. This indicates that the strong interaction of Stat_3, the phosphorylated peptide, is attributed to its phosphoserines. DEWA_5, the fusion construct of the truncated YaaD protein, the mineralizing P11-4 peptide and the hydrophobin DEWA showed good binding abilities as well as an enhanced nucleation in artificial saliva. The amphiphilic hydrophobin DEWA belongs to the class I of hydrophobins, which is able to form stable monolayers containing laterally arranged fibers.58 They are known to adsorb strongly to hydrophobic and hydrophilic surfaces and their high surface activity have led to applications in surface modifications to coatings for drug nanoparticles.59–63 Recently, a fusion construct of hydrophobin HFB II and a calcium binding protein sequence has been used for the mineralization of calcium carbonate.64 We observed good binding abilities to hydroxyapatite in the mouthwash formulation regarding the construct DEWA_2 (40aaYaaD-DEWA). By inserting a hydroxyapatite binding peptide the binding could be further increased as in the case of DEWA_4 (40aaYaaD-CPL12DEWA) and DEWA_5 (40aaYaaD-P11-4-DEWA). These two constructs revealed, in addition to their binding activity, an enhanced nucleation activity. Neither DEWA_2 nor only the peptide P11-4 showed an effect in nucleation, whereas the fusion construct DEWA_5 exhibits a strong increase in mineral formation. P11-4 is a rationally designed peptide. Above a concentration of 15 mg/mL this peptide is able to self-assemble into a fibrillar network serving as a template for nucleation.12 In our experiments the concentration of the peptide P11-4 was much lower (25 µM corresponds to a concentration of 0.04 mg/mL) and no self-assembly of the peptide was observed, which might be the reason why no increase in nucleation was observed. This demonstrates the importance of having a rigid scaffold to enhance nucleation. Many proteins exhibit a mineralizing activity after immobilization on a solid scaffold or after self-assembly.8–11,65–67 In solution, these proteins failed to mineralize hydroxyapatite. In our work the stabilization of P11-4 in the construct DEWA_5 was not observed by self-assembly of the peptide, but achieved by fusion to DEWA and YaaD. Beside binding abilities this fusion protein might be also acting as a scaffold.


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The peptide P11-4 was rationally designed to self-assemble. Through π- π interactions the aromatic amino acids phenylalanine and tryptophan trigger the self-assembly. Also the terminal glutamine residues contribute to the self-assembly by hydrophobic interactions.12,68 Therefore, it is perhaps not surprising that deletion of the terminal glutamine residues does not affect the nucleation behavior as they are only introduced into the peptide sequence to trigger the self-assembly. That acidic amino acid residues (E53, E55, E57) play a key role in biomineralization processes has been widely described.9,65,69,70 The alanine scan and also the deletion variants of DEWA_5 support this. By changing the glutamate residues into alanine or deleting these residues a decrease in nucleation activity was observed. On the other hand, the substitution of the glutamate residues within the truncated variant ∆3 by aspartate led to higher binding ability. This is consistent with other experiments found in literature which show that aspartate residues have a higher binding affinity towards hydroxyapatite then glutamate residues.9,69,71,72 Additionally, the fusion construct DEWA_5 could be further engineered towards a faster mineral formation. The engineered variant DEWA_5a contains the sequence (SD)3. By comparing this sequence to proteins that are naturally involved in the formation of hydroxyapatite the similarity to the motif D-SS in the dentin phosphoprotein is obvious. Beside the predominate motif D-S-S, also repeats of (DS) were found in the sequence of DPP.25,73 The further optimization of the nucleation activity of the original peptide P11-4 embedded in the 40aa Yaad-DEWA construct (DEWA_5) led to an amino acid motif which evolved in the biomineralization of hydroxyapatite. It is noteworthy to point out that the serin residues in the wildtype dentin phosphoprotein are phosphorylated, whereas the serin residues in DEWA_5a are unphosphorylated. Although phosphorylation plays an important role in vivo biomineralization of hydroxyapatite by this motif, Yarbrough and coworkers could show that the unphosphorylated (DSS)n-motif inherits also a nucleation activity when being conformational stabilized. This was shown by the immobilization of (DSS)8 onto polystyrene beads.9 The engineered motif (SD)3 is embedded in the 40aa YaaD-DEWA construct and thereby in a conformational less flexible state than as the peptide alone in solution. Inserting the predominate motif (DSS)n into the 18 ACS Paragon Plus Environment

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40aa Yaad-DEWA construct (DEWA_2) would be interesting as we already observed a higher nucleation rate with the motif (SD)3. The original peptide P11-4 was designed to mimic proteins of extracellular matrices like in nature.12 Within the fusion protein (DEWA_5) this sequence was subsequently modified into an amino acid motif (SD)3 that is found in nature.

Conclusions The work described here in has several significant implications in protein/peptide assisted biomineralization. First, the study has identified fusion proteins that are able to bind to and nucleate hydroxyapatite. Strong binding affinity to hydroxyapatite is not necessarily an indication of its nucleation activity. Secondly, certain amino acid residues were found to play a role in the binding ability and nucleation of hydroxyapatite. We could demonstrate that non-aromatic, hydroxylated (S) and charged (R/D) amino acid residues show beneficial effects in the binding and nucleation of hydroxyapatite. The sequence responsible for the nucleation resembled the tripeptide repeat (D-S-S) of dentin phosphoprotein, one of the major non-collagenous proteins involved in the mineralization of dentin. Starting with a rationally designed peptide sequence (P11-4) we have engineered a unique hydroxyapatite-binding motif (SD)3 with biological nucleation activity within a fusion protein.

Supporting Information The data for the nucleation of the statherin variants, alanine scan single variants and variants lacking glutamine residues in the sequence of P11-4 within DEWA_5. This material is available free of charge via the Internet at http://pubs.acs.org

Acknowledgements We would like to acknowledge BASF SE, Ludwigshafen, Germany for their financial support. We especially thank Dr. Nina Schneider, Dr. Stefan Jenewein and Dr. Claus Bollschweiler for many helpful discussions. We are grateful to Moritz Schmelz, Institute of Mineralogy, University of Stuttgart for the 19 ACS Paragon Plus Environment

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preparation of the tooth slices. We also thank Dr. Thomas Theye, Institute of Mineralogy, University of Stuttgart for the XRD measurements and Dr. Stephan Nußberger and Dr. Michael Schweikert of the Institute of Biology, University Stuttgart for the TEM measurements.

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61. Scholtmeijer, K.; Janssen, M. I.; Gerssen, B.; de Vocht, M. L.; van Leeuwen, B. M.; van Kooten, T. G.; Wösten, H. A.; Wessels, J. G. Surface modifications created by using engineered hydrophobins. Appl. Environ. Microbiol. 2002, 68, 1367-1373. 62. Janssen, M. I.; van Leeuwen, M. B.; Scholtmeijer, K.; van Kooten, T. G.; Dijkhuizen, L.; Wösten, H. A. Coating with genetic engineered hydrophobin promotes growth of fibroblasts on a hydrophobic solid. Biomaterials 2002, 23, 4847-4854. 63. Hektor, H. J.; Scholtmeijer, K. Hydrophobins: Proteins with potential. Curr. Opin. Biotechnol. 2005, 16, 434-439. 64. Heinonen, H.; Laaksonen, P.; Linder, M. B.; Hentze, H. Engineered hydrophobin for biomimetic mineralization of functional calcium carbonate microparticles. J. Biomater. Nanobiotechnol. 2014, 5, 1-7. 65. He, G.; George, A. Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro. J. Biol. Chem. 2004, 279, 11649-11656. 66. Lee, J.; Choo, J.; Choi, Y.; Park, J.; Min, D.; Lee, S.; Rhyu, H. K.; Jo, I. H.; Chung, C. P.; Park, Y. J. Assembly of collagen-binding peptide with collagen as a bioactive scaffold for osteogenesis in vitro and in vivo. Biomaterials 2007, 28, 4257-4267. 67. Fujisawa, R.; Kuboki, Y.; Sasaki, S. Changes in interaction of bovine dentin phosphophoryn with calcium and hydroxyapatite by chemical modifications. Calcif. Tissue Int. 1986, 39, 248251. 68. Aggeli, A.; Bell, M.; Carrick, L. M.; Fishwick, C. W. G.; Harding, R.; Mawer, P. J.; Radford, S. E.; Strong, A. E.; Boden, N. pH as a trigger of peptide beta-sheet self-assembly and reversible switching between nematic and isotropic phases. J. Am. Chem. Soc. 2003, 125, 9619-9628. 69. Hunter, G. K.; Goldberg, H. A. Nucleation of hydroxyapatite by bone sialoprotein. Proc. Natl. Acad. Sci. USA 1993, 90, 8562-8565. 70. Weiner, S.; Addadi, L. Design strategies in mineralized biological materials. J. Mater. Chem. 1997, 7, 689-702. 71. Fujisawa, R.; Wada, Y.; Nodasaka, Y.; Kuboki, Y. Acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals. Biochim. Biophys. Acta 1996, 1292, 53-60. 72. Hunter, G. K.; Kyle, C. L.; Goldberg, H. A. Modulation of crystal formation by bone phosphoproteins: structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation. Biochem. J. 1994, 300, 723-728. 73. Sabsay, B.; Stetler-Stevenson, W. G.; Lechner, J. H.; Veis, A. Domain structure and distribution in dentin phosphophoryn. Biochem. J. 1991, 276, 699-707.


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Statherins

Hydrophobins

Antifreeze proteins (AFP)

Amelogenin

Table 1: Proteins, fusion constructs and peptides used in this study.

Controls

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Name

Proteins, fusion constructs and peptides

Species

Amelo1

Amelogenin*

Homo sapiens

17.4

Amelo2

Leucine rich Amelogenin protein*

Homo sapiens

8.5

Amelo3

Amelogenin-DEWA*

Homo sapiens / Aspergillus nidulans

30.6

Amelo4

Amelogenin-Perlucin*

Homo sapiens / Haliotis laevigata

35.6

Amelo5

Amelogenin (∆42 aa N-term., ∆17 aa C-term.)-DEWA*

Amelo6

Amelogenin (23 aa C-term.)-DEWA*

Homo sapiens / Aspergillus nidulans Homo sapiens / Aspergillus nidulans

MW [kDa]

22.9 16.8

Amelo7

Amelogenin (M11A, P21A)*

Homo sapiens

17.0

Amelo8

MGKK-CPL12 (QPYHPTIPQSVH)54-G3Amelogenin (M11A, P21A)* $

Homo sapiens

19.0

Amelo9

R5 (SSKKSGSYSGSKGSKRRIL)53-Amelogenin*

Homo sapiens

19.4

AFP_1

AFP 4*

Choristoneura fumiferana

10.0

AFP_2

CherryTM tag-AFP Type III*°

Macrozoarces americanus

19.9

AFP_3

Carboanhydrase (CanA)-AFP 9*

Myceliophthora thermophile / Lolium perenne

40.0

AFP_4

AFP 9*

Lolium perenne

12.6

AFP_5

AFP 3*

Myoxocephalus octodecemspinosus

13.1

AFP_6

AFP 4 (68 aa C-term.)*


6.9

AFP_7

HA4 (IPTLPSS)-G3-AFP 4*$#


11.0

AFP_8

AFP 4 (T-->S mult)*§


10.8

AFP_9

CherryTM tag- AFP (isoform 4-9)*°

Tenebrio molitor

23.8

Bacillus subtilis / Aspergillus nidulans Bacillus subtilis / Aspergillus nidulans Bacillus subtilis / Aspergillus nidulans Bacillus subtilis / Aspergillus nidulans Bacillus subtilis / Aspergillus nidulans

DEWA_1

YaaD-DEWA*48

DEWA_2

40aa YaaD-DEWA *48

DEWA_3

R5 (SSKKSGSYSGSKGSKRRIL)53-40aa YaaD-DEWA*

DEWA_4

40aa YaaD-Xa-MGKK-CPL12 (QPYHPTIPQSVH)54DEWA*

DEWA_5

40aa YaaD-Xa-MGKK-P11-4 (QQRFEWEFEQQ)12-DEWA*

Stat_1

Viral Protein 1-100aa Statherin-DD*$

Polyomavirus / Homo sapiens

18.3

Stat_2

Statherin-peptid unphosphorylated

Homo sapiens (synthetic)

2.3

Stat_3

Statherin-peptid phosphorylated

Homo sapiens (synthetic)

2.5

Stat_4

Statherin-peptid unphosphorylated-G3-AFP4*$


12.7

MI tooth mousseTM

Peptides from MI tooth mousseTM 49

synthetic

1.3

(DSS)6

(DSS)6 9

synthetic

1.8

#3-1

Reference peptide #3-1 (LIKHILHRL)9

synthetic

1.1

*: harboring a C-terminal His6-tag $ : G: glycine spacer; D: aspartate spacer § : contains multiple substitutions of threonine by serine # : HA4: hydroxyapatite binding peptide, unpublished data


47.0 19.0 21.0 20.7 21.8

Biomacromolecules

TM

°: Cherry tag, Delphi Genetics SA, Charleroi, Belgium Xa: Factor Xa protease cleavage site (IEGR)

Table 2: Results of the binding and nucleation experiments. Binding mouthwash

Amelo1

22±5

-

o

Amelo2

59±1

-

o

Amelo3

56±1

-

o

Amelo4

60±1

-

o

Amelo5

58±5

-

o

Amelo6

62±3

-

+

Amelo7

69±2

-

o

Amelo8

60±12

-

o

Amelo9

12±2

-

+

AFP_1

33±7

-

+

AFP_2

59±13

-

+

AFP_3

80±7

-

o

AFP_4

45±10

-

+

AFP_5

60±8

-

+

AFP_6

66±6

-

o

AFP_7

59±1

6±3

o

AFP_8

57±2

16±3

o

AFP_9

91±1

3±2

o

Hydrophobins

HBS-T

DEWA_1

30±5

-

o

DEWA_2

43±11

25±3

o

DEWA_3

63±1

2±1

-

DEWA_4

80±3

36±2

+

DEWA_5

72±1

30±1

+

Statherins

Antifreeze proteins

Amelogenin

Nucleation

Stat_1

75±2

-

-

Stat_2

16±4

-

o

Stat_3

86±2

14±3

-

Controls

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Stat_4

73±3

2±1

-

MI tooth mousseTM

70±9

30±1

-

(DSS)6

84±9

-

o

#3-1

-

-

o

-: delayed nucleation; o: same nucleation trend; +: enhanced nucleation


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Biomacromolecules

Figure 1: Binding of fluorescence labeled proteins to human tooth slices in the mouthwash formulation. Bright field images of slices treated with the positive control MI tooth mousseTM (a), the negative control #3-1 (c), DEWA_4 (e) and DEWA_5 (g). Fluorescence images of the shown bright field for MI tooth mousseTM (b), #3-1 (d), DEWA_4 (f) and DEWA_5 (h). The tooth hard tissues enamel (E) and dentin (D) are marked by the white letters. Scale bars correspond to 500 µm.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a)

b)

Figure 2: Nucleation in artificial saliva. Calcium consumption in the presence of fusion proteins containing either antifreeze proteins (a) or hydrophobins (b) with the control without protein.


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Biomacromolecules

Figure 3: X-ray powder diffraction pattern of the mineral formed in the presence of either the hydrophobin fusion proteins DEWA_5 (a), and DEWA_4 (b) or no protein (c); (d) standard pattern of hydroxyapatite (JCPDS card No. 09-0432).

Figure 4: TEM images of the formed mineral. All samples show plate-like crystals. Crystals are spherulite arranged in the sample without protein (a). In DEWA_4 (b) and DEWA_5 (c) the crystals show a more bundle-like-fibrous arrangement. Bars correspond to 200 nm.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5: Nucleation in artificial saliva. Calcium consumption in the presence of the peptide P11-4 and DEWA_2 without an inserted peptide.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 6: a) Schematic scheme of the construct DEWA_5, the alanine variants and the truncated variants. The amino acid sequences of the full length P11-4 (DEWA_5), the alanine substituted and truncated peptides are shown between the truncated synthase YaaD and the hydrophobin DEWA protein. Alanine substitutions are bold and underlined. b) Schematic scheme of the focused library variants based on the sequence of the truncated variant ∆3. Substitutions are bold and underlined, X = S, Y, R, H, V; Z = Q, D. 31 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7: Nucleation in artificial saliva. Calcium consumption in the presence of DEWA_5 and variants lacking glutamate residues in the sequence of P11-4 within DEWA_5 either by alanine substitution (EE53/55AA and EEE53/55/57AAA) or by deletion (∆4 and ∆5).

Figure 8: Nucleation in artificial saliva. Calcium consumption in the presence of DEWA_5 and the variants of the focused library based on the truncated sequence FEWEFE of P11-4 within DEWA_5.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 9: Nucleation in artificial saliva. Calcium consumption in the presence of DEWA_5, the truncated variant ∆3 and the engineered variants EEE50/52/54DDD, DEWA_5a, DEWA_5b and a control without protein.

Figure 10: Characterization of the mineral formed during the nucleation in artificial saliva with the variants DEWA_5a (a), DEWA_5b (b), and EEE50/52/54DDD (c) compared with the standard pattern of hydroxyapatite (JCPDS card No. 09-0432) (d).


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 11: Quantitative binding results of DEWA_5 and the variants EEE50/52/54DDD, DEWA_5a and DEWA_5b to hydroxyapatite in HBS-T buffer (white bars) and the mouthwash formulation (grey bars).


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Biomacromolecules

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