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AcceleratedNucleationofHydroxyapatiteUsinganEngineeredHydrophobinFusionProtein

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Accelerated Nucleation of Hydroxyapatite Using an EngineeredHydrophobin Fusion ProteinMelanie Melcher,† Sandra J. Facey,† Thorsten M. Henkes,† Thomas Subkowski,‡ and Bernhard Hauer*,†

†Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany‡Fine Chemicals Research, BASF SE, 67056 Ludwigshafen, Germany

*S Supporting Information

ABSTRACT: Calcium phosphate mineralization is of particularinterest in dental repair. A biomimetic approach using proteins orpeptides is a highly promising way to reconstruct eroded teeth. Inthis study, the screening of several proteins is described for theirbinding and nucleating activities toward hydroxyapatite. Out of 27tested candidates, only two hydrophobin fusion proteins showedbinding abilities to hydroxyapatite in a mouthwash formulation andan increased nucleation in artificial saliva. Using a semirationalapproach, one of the two candidates (DEWA_5), a fusion proteinconsisting of a truncated section of the Bacillus subtilis synthaseYaaD, the Aspergillus nidulans hydrophobin DEWA, and therationally designed peptide P11-4 described in the literature, couldbe further engineered toward a faster mineral formation. Thevariants DEWA_5a (40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA) were able to enhance thenucleation activity without losing the ability to form hydroxyapatite. In the case of variant DEWA_5b, an additional increase inthe binding toward hydroxyapatite could be achieved. Especially with the variant DEWA_5a, the protein engineering of therationally designed peptide sequence resulted in a resemblance of an amino acid motif that is found in nature. The engineeredpeptide resembles the amino acid motif in dentin phosphoprotein, one of the major proteins involved in dentinogenesis.

■ INTRODUCTION

The mammalian tooth is composed of several tissues. One ofthem is enamel, the hardest material in the vertebrate body.1

Enamel contains a high amount of hydroxyapatite, approx-imately 95%. In enamel, the hydroxyapatite nanocrystals arehighly organized and elongated in the direction of the c-axis.2

The extreme hardness and stability of the dental enamel isattributed to this special structure. Though, because of the highcontent of inorganic material and the absence of organiccompounds, like polymers and proteins, there is no in vivomechanism to reconstruct the dental enamel once destroyed.Required is an approach to form enamel-like material in asynthetic manner. In the past, many research groups havefocused on the in vitro synthesis of enamel.1,3−12 Protein orpeptide mediated biomineralization has been found to be apromising method to reconstruct enamel because it can occurunder physiological conditions to provide a biocompatiblematerial. Detailed studies of extracellular matrix proteins duringthe development of enamel or dentin have led to a deeperunderstanding of the biomineralization process of teeth.8,9,13−15

The major proteins amelogenin and dentin phosphoproteinhave been found to play an important role in modulating themineralization of organized calcium phosphate crystals.Amelogenin is the major protein component of the enamelextracellular matrix during enamel formation. The self-assemblyof amelogenin into nanospheres is believed to play a key role in

controlling the oriented and elongated growth of thehydroxyapatite crystals.16,17 In the case of dentinogenesis,nucleation and growth of hydroxyapatite is mainly attributed tononcollagenous proteins that are rich in acidic amino acids.18

One of these noncollagenous proteins of the dentinextracellular matrix is the dentin phosphoprotein (DPP). Itcontains an unusually high amount of phosphorylated serineresidues and aspartic acids with approximately 90%.19 In thepresence of Ca2+ ions, DPP self-assembles into a β-sheet-likeconformation, serving as a high negatively charged surface. Thissurface may not only interact with the growing hydroxyapatitecrystal, but also be involved in the first crystal deposition duringdentinogenesis.20−22 Also, in vitro experiments have demon-strated the mineralizing effect of immobilized DPP.23,24 Theseabilities may be attributed to the predominate motif D-S-S inDPP.25 Yarbrough et al.9 have further demonstrated the bindingabilities of peptides containing a different number of thepredominant motif D-S-S (DSS)n without phosphorylation tohydroxyapatite and mineralized tissues. They also showed themineralizing effect of (DSS)8 after immobilizing ontopolystyrene beads, whereas in free solution these peptidesfailed to enhance mineralization. Also, the unphosphorylated

Received: January 27, 2016Revised: March 21, 2016

Article

pubs.acs.org/Biomac

© XXXX American Chemical Society A DOI: 10.1021/acs.biomac.6b00135Biomacromolecules XXXX, XXX, XXX−XXX

triple repeat (DSS)3 promotes apatite deposition on deminer-alized enamel after binding of the peptide to eroded teeth.11

Based on the knowledge that these proteins present favorablesites for nucleation in ordered and rigid conformation, therehave been approaches to mimic the extracellular matrix (ECM)of such systems by the rational design of peptides.12,26−28 Oneexample is the rationally designed self-assembling peptide P11-4.12 This anionic peptide can self-assemble into β-sheet latticesthat further assemble into a fibrillar network. These networksserve as a scaffold for crystal deposition like ECM does innature. Furthermore, Kirkham et al.12 demonstrated aremineralization effect of this peptide in dental lesions.For clinical applications or oral care products, different

approaches have been developed to treat eroded enamel orcaries lesions. Fluoride was identified to be an effectiveremineralizing agent.29 This was due to the substitution ofhydroxyl groups by fluoride ions in which fluorapatite(Ca10(PO4)6F2) is formed. This shows a higher stability andis insoluble in a broader pH range.30−32 Nanohydroxyapatiteshows a crystal structure similar to that of dental apatite.33,34

Many efforts demonstrated that nanosized hydroxyapatiteshows a positive effect on the remineralization of the erodedenamel and caries lesions.33,35 Furthermore, phosphopeptidesderived from casein were applied in combination with

amorphous calcium phosphate (CPP-ACP) in oral careproducts.36 In clinical studies, the remineralizing potential ofCCP-ACP was demonstrated.32,37 But these approaches alsoinherit problems. There is still a requirement to improve thedelivery of fluoride in optimal amounts to overcome high riskcaries in many individuals.38 An excessive uptake may result inenamel fluorosis, especially in children’s teeth.39−41 Abiomimetic approach would lead to a direct reconstruction ofthe decayed enamel structure.This study seeks to identify and characterize proteins that are

able to remineralize hydroxyapatite in vitro. Therefore, a panelof various fusion proteins was characterized in regard to theirbinding and mineralization abilities. The panel resulted fromfour different proteins, which were used in their native form, ascleavage products or as fusion proteins, in combination withliterature described peptides. Amelogenin and statherin werechosen because of their described binding activities tohydroxyapatite,42,43 whereas antifreeze proteins and theAspergillus nidulans hydrophobin DEWA (formerly known asCAN4)44 were chosen because of their well-known surfaceactive abilities.45−47 DEWA was also previously reported to beexpressible in E. coli when being expressed as a fusion proteinwith the Bacillus subtilis synthase YaaD, which allows its usagein industrial applications.48 All tested hydrophobin DEWA-

Table 1. Proteins, Fusion Constructs, and Peptides Used in This Study

name proteins, fusion constructs, and peptides speciesMW(kDa)

amelogenin Amelo1 amelogeninb Homo sapiens 17.4Amelo2 leucine-rich amelogenin proteinb Homo sapiens 8.5Amelo3 amelogenin-DEWAb Homo sapiens/Aspergillus nidulans 30.6Amelo4 amelogenin-perlucinb Homo sapiens/Haliotis laevigata 35.6Amelo5 amelogenin (Δ42 aa N-term., Δ17 aa C-term.)-DEWAb Homo sapiens/Aspergillus nidulans 22.9Amelo6 amelogenin (23 aa C-term.)-DEWAb Homo sapiens/Aspergillus nidulans 16.8Amelo7 amelogenin (M11A, P21A)b Homo sapiens 17.0Amelo8 MGKK-CPL12 (QPYHPTIPQSVH)54-G3-amelogenin

(M11A, P21A)b,cHomo sapiens 19.0

Amelo9 R5 (SSKKSGSYSGSKGSKRRIL)53-amelogeninb Homo sapiens 19.4antifreeze proteins(AFP)

AFP_1 AFP 4b Choristoneura fumiferana 10.0AFP_2 Cherry tag-AFP type IIIb,f Macrozoarces americanus 19.9AFP_3 carboanhydrase (CanA)-AFP 9b Myceliophthora thermophile/

Lolium perenne40.0

AFP_4 AFP 9b Lolium perenne 12.6AFP_5 AFP 3b Myoxocephalus octodecemspinosus 13.1AFP_6 AFP 4 (68 aa C-term.)b Choristoneura fumiferana 6.9AFP_7 HA4 (IPTLPSS)-G3-AFP 4b,c,e Choristoneura fumiferana 11.0AFP_8 AFP 4 (T→S mult)b,d Choristoneura fumiferana 10.8AFP_9 Cherry tag-AFP (isoform 4−9)b,f Tenebrio molitor 23.8

hydrophobins DEWA_1 YaaD-DEWAb,48 Bacillus subtilis/Aspergillus nidulans 47.0DEWA_2 40aa YaaD-DEWAb,48 Bacillus subtilis/Aspergillus nidulans 19.0DEWA_3 R5 (SSKKSGSYSGSKGSKRRIL)53-40aa YaaD-DEWAb Bacillus subtilis/Aspergillus nidulans 21.0DEWA_4 40aa YaaD-Xa-MGKK-CPL12 (QPYHPTIPQSVH)54-DEWAa,b Bacillus subtilis/Aspergillus nidulans 20.7DEWA_5 40aa YaaD-Xa-MGKK-P11-4 (QQRFEWEFEQQ)12-DEWAa,b Bacillus subtilis/Aspergillus nidulans 21.8

statherins Stat_1 viral protein 1-100aa statherin-DDb,c polyomavirus/Homo sapiens 18.3Stat_2 statherin-peptid unphosphorylated Homo sapiens (synthetic) 2.3Stat_3 statherin-peptid phosphorylated Homo sapiens (synthetic) 2.5Stat_4 statherin-peptid unphosphorylated-G3-AFP4b,c Choristoneura fumiferana 12.7

controls MI toothmousse

peptides from MI tooth mousse49 synthetic 1.3

(DSS)6 (DSS)69 synthetic 1.8

#3-1 reference peptide #3-1 (LIKHILHRL)9 synthetic 1.1aXa: Factor Xa protease cleavage site (IEGR). bHarboring a C-terminal His6-tag.

cG: glycine spacer; D: aspartate spacer. dContains multiplesubstitutions of threonine by serine. eHA4: hydroxyapatite binding peptide, unpublished data. fCherry tag, Delphi Genetics SA, Charleroi, Belgium.

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B

constructs contained an amino-terminal fusion to the Bacillussubtilis synthase YaaD or a truncated version consisting of 40amino acids of YaaD, as described as H*protein A or B byWohlleben and co-workers, respectively.48

In this study, we investigated the ability of the differentfusion proteins and peptides to bind to hydroxyapatite in HBS-T buffer and in a mouthwash formulation. Furthermore,nucleation activities of these proteins were analyzed andcharacterized using X-ray diffraction and transmission electronmicroscopy. For a more detailed determination of the crucialamino acids needed for nucleation, our best candidate, a fusionconstruct of the peptide P11-4 and the fusion protein consistingof hydrophobin DEWA and the truncated version of YaaD, wasfurther investigated by site-directed mutagenesis. Based on thesequence of P11-4, the nucleation activity was enhanced bydifferent mutagenesis strategies.

■ EXPERIMENTAL SECTIONMaterials. The fusion proteins consisted of nine amelogenin

constructs, nine antifreeze constructs, five hydrophobin constructs, andfour statherin variants. Peptides extracted out of commercially availableMI tooth mousse,49 (DSS)6

9 and peptide #3−1 (LIKHILHRL),9

which was described as a nonbinder toward hydroxyapatite, were usedas controls. All 30 proteins and peptides were obtained purified or aslyophilized powder, respectively, from the BASF SE (Ludwigshafen,Germany; Table 1).Bacterial Strains and Culture Conditions. E. coli XL1-blue was

used for routine subcloning and plasmid propagation. The E. coli strainM15 [pREP4]50 was used for expression of the recombinant proteinsin the vector pQE60 (Qiagen, Hilden, Germany). Media preparationand bacterial manipulations were performed according to standardmethods.51 Where appropriate, ampicillin (100 μg/mL, finalconcentration) and kanamycin (25 μg/mL, final concentration) wereadded to the medium.Construction of Hydrophobin Variants Based on DEWA_5.

The construct DEWA_5 containing the sequence encoding for40aaYaaD-P11-4-DEWA, consisting of the first 40 amino acid residuesof the synthase YaaD of Bacillus subtilis for recombinant expression inE. coli,48 the 11-mer hydroxyapatite binding peptide P11-4

12 andhydrophobin DEWA, was kindly provided by BASF. For the alaninescan, the codons for the amino acids of the P11-4 peptide sequence,QQRFEWEFEQQ, between amino acid positions 49 and 59 inDEWA_5 were exchanged to codons for alanine by site-directedmutagenesis using the QuikChange method (Stratagene). To identifywhich part of the P11-4 sequence is important for nucleation deletionvariants were constructed. Based on the truncated variant Δ3(40aaYaaD-FEWEFE-DEWA), a small focused library was created.Negatively charged glutamate residues were exchanged either toaspartate 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. colistrain M15 [pREP4].50 A single colony was cultured overnight in 3 mLof 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 × 7H2O,0.7 mM CaCl2 × 2H2O, 10 mL/L 5× 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/LFeSO4 × H2O, 50 mg/L ZnSO4 × 7 H2O, 15 mg/L MnCl2 × 4 H2O,150 mg/L H2BO3, 100 mg/L CoCl2 × 6 H2O, 5 mg/L CuCl2 × 2H2O, 10 mg/L NiCl2 × 6 H2O, and 15 mg/L Na2MoO4 × 2 H2O.Overnight cultures were diluted to an OD600 of 0.5 in fresh EC3 mediacontaining the appropriate antibiotics and cultivated at 37 °C, 180 rpmfor 90 min. Expression of the recombinant proteins was induced withisopropyl-ß-D-thiogalactopyranoside (IPTG) to a final concentration

of 0.5 mM. After additional incubation for 3 h at 37 °C, 180 rpm thecells were harvested by centrifugation for 20 min, 6000 rpm at 4 °C.

The DEWA_5 variants were purified by renaturing the inclusionbodies. Therefore, cells were disrupted by sonification, washed withTBS buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5), and centrifuged(10 min, 8000 rpm, 4 °C). The supernatant was discarded and theobtained inclusion bodies were renaturated by the addition of NaOHto a final concentration of 0.1 M and stirring for 10 min at roomtemperature. The pH was adjusted to 7.5 by titration with 2% H3PO4and insoluble proteins were separated by centrifugation (20 min, 8000rpm, 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 DNAwas removed by a treatment with DNase I for 1 h at roomtemperature. For nucleation, proteins were again dialyzed against 5mM Tris-HCl, pH 7.5. Protein expression was analyzed by 15%sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). Gels were stained with Coomassie Brilliant Blue (R 250).

Protein Labeling. For binding studies 5(6)-carboxyfluorescein wasconjugated to the N-terminus of the proteins by using thecorresponding succinimidyl ester (Invitrogen, Darmstadt, Germany).The labeling reaction was performed according to the manufacturer’sprotocol (Invitrogen, Darmstadt, Germany). After conjugation, thelabeled protein was purified either with a PD-10 desalting column forproteins larger than 5 kDa or a PD MidiTrap G-10 for smaller proteins(GE Healthcare, Buckinghamshire, U.K.). Protein concentration wasdetermined by the bicinchoninic acid method using the BCA ProteinAssay Kit (Thermo Scientific, Dreieich, Germany).

Binding to Hydroxyapatite Powder. For the binding experi-ments N-terminal labeled proteins were used in a final concentrationof 2 μM. The binding was carried out in HBS-T buffer (150 mMNaCl, 50 mM HEPES, pH 7.5, 0.1% Tween 20) and a mouthwashformulation (6.0 mM NaF, 0.5 mM sodium saccharine, 16.7 mMNaH2PO4, 28.1 mM Na2HPO4, 407.2 mM glycerol, 492.8 mMpropylene glycol, 150.9 mM sorbitol, 0.5% Poloxamer 407). Theproteins were exposed to 10 mg hydroxyapatite powder (Fluka, Basel,Switzerland) for 2 h at 37 °C. After five wash steps with HBS-T buffer,the bound protein was visualized by fluorescence microscopy with theappropriate filters (excitation 475/50; emission 525/50). To calculatethe amount of bound protein, the fluorescence intensity of the solutionwas measured before (Fi) and after (Ff) the binding reaction withFluostar Galaxy (BMG, Ortenberg, Germany). The amount of boundprotein cbound was calculated according to the equation: cbound = [1 −(Ff/Fi)] × c0, where c0 is the initial protein concentration. The peptide#3-1 (LIKHILHRL), which was described as a nonbinding peptide tohydroxyapatite, was used as a negative control.9 All bindingexperiments were carried out as triplicates.

Binding to Human Teeth Slices. The binding of the fusionproteins to human teeth in the mouthwash formulation was alsoinvestigated. Adult human teeth were sagittal sectioned into slices, 100μm in thickness, using a microtome (Institute of Mineralogy,University of Stuttgart). Prior to the use, the tooth slices were rinsedwith deionized water and equilibrated for 30 min in mouthwashformulation. After incubation for 1 h in mouthwash formulationcontaining 2 μM of labeled protein, the tooth slices were rinsed againwith deionized water and imaged by fluorescence microscopy.

Nucleation Assays. To determine the nucleating abilities of theproteins the decrease in Ca2+ concentration was measured. Nucleationwas carried out in artificial saliva52 containing 8.7 mM KCl, 0.6 mMMgCl2, 1.5 mM CaCl2, 4.6 mM K2HPO4, 2.7 mM KH2PO4, and 25μM protein in a final volume of 1 mL. As a negative control, only thenucleation solution without any protein was used. All solutions werefiltered (0.2 μm) prior to use to avoid uncontrolled nucleation due tosmall particles. Periodically, 30 μL samples were taken every hour for atotal of 5 h, centrifuged 2 min, 13000 rpm, and the calciumconcentration of the supernatant was determined. For the calciumdetection, 30 μM o-cresolphthalein, 2.7 mM 8-hydroxyquinoline, 20mM 2-amino-2-methyl-1-propanol, pH 10.5, and 20 μL of the samplewere mixed and the absorbance was monitored at 575 nm using aSpectramax (Molecular Devices, Biberach, Germany). All nucleationexperiments were carried out at 37 °C in triplicates.

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X-ray Powder Diffraction. For characterization of the hydrox-yapatite crystals, the nucleation assay was scaled up to a final volume of50 mL. The formed precipitate after 4 h was centrifuged and rinsedfive times with deionized water to remove remaining ions. The mineralwas dried and characterized by X-ray powder diffraction analysis usinga Bruker D8 Advance diffractometer of Cu Kα radiation at 1.54 Å witha scanning rate of 0.015°/step with 2θ in the range from 24° to 67°.The experimental patterns obtained were compared with ahydroxyapatite standard (card No. 09−0432) compiled by the JointCommittee on Powder Diffraction and Standards (JCPDS).Transmission Electron Microscopy (TEM). Nucleation was

stopped after 4 h by centrifugation of the nucleation mixture. Themineral was rinsed five times with deionized water and resuspended inwater. A 5 μL sample was applied to a Formvar/carbon-coated Cu100-mesh TEM grid (Plano, Wetzlar, Germany) and air-dried. Imagingwas performed using a Tecnai G2 Sphera TEM at 120 kV.

■ RESULTS

Selection of Hydroxyapatite-Binding Proteins. A panelof fusion proteins containing amelogenin, statherin, antifreezeproteins, or hydrophobin was screened for their bindingactivities to hydroxyapatite in HBS-T buffer and a mouthwashformulation. The fusion proteins were labeled with 5(6)-carboxyfluorescein and exposed to hydroxyapatite powder. Thequantitative binding results are summarized in Table 2.

Binding to hydroxyapatite was detected in all fusion proteinsin HBS-T buffer, except for the negative control peptide #3-1(LIKHILHRL), which was described in the literature9 as anonbinding peptide for hydroxyapatite. Both positive controls,MI tooth mousse49 and (DSS)6,

9 showed good binding withmore than 70% bound peptide. In the group of the amelogeninderivatives the amount of bound protein to hydroxyapatite wasin the range of 60% except for the full length amelogenin(Amelo_1) and the fusion construct consisting of the titaniamineralizing peptide R5 (SSKKSGSYSGSKGSKRRIL)53

(Amelo_9). Both constructs, Amelo_1 and Amelo_9, showedonly minor binding activity in HBS-T buffer. In the case of theantifreeze fusion proteins, binding was detected in the range of34% for the antifreeze protein 4 from Choristoneura fumiferana(AFP_1) and 91% for the antifreeze protein from Tenebriomolitor (AFP_9) with the fusion partner Cherry tag (DelphiGenetics SA, Charleroi, Belgium). Also, the hydrophobinconstructs showed good binding abilities between 30% forDEWA_1, consisting of the full length Bacillus subtilis synthaseYaaD and the hydrophobin DEWA from Aspergillus nidulansand 80% for the fusion construct of a truncated version ofYaaD, the hydroxyapatite binding peptide CPL12 (QPYH-PTIPQSVH)54 and DEWA (DEWA_4). The group ofstatherin fusion proteins revealed good binding to hydrox-yapatite with more than 70% bound protein except Stat_2, theunphosphorylated statherin peptide, with only 16%.A completely different binding behavior for the fusion

proteins was observed when the HBS-T buffer was exchangedwith the mouthwash formulation. The positive control MItooth mousse revealed binding to hydroxyapatite of only about30%. In contrast, the other positive control, (DSS)6 lost itsbinding activity completely. Also, in the amelogenin group, nobinding to hydroxyapatite was detected. For the antifreezefusion proteins, a similar behavior could be observed. Out ofthe nine proteins, only two showed moderate binding towardhydroxyapatite in the mouthwash formulation (AFP_7,AFP_8). AFP_9, the antifreeze protein from T. molitor,which had excellent binding capabilities in HBS-T bufferrevealed only a minor activity (3%) in the mouthwashformulation. In the group of the statherin variants, thephosphorylated Stat_3 showed moderate binding to hydrox-yapatite with about 14%. In contrast, the fusion proteinscontaining hydrophobin kept their binding ability. OnlyDEWA_1, consisting of the full length YaaD and thehydrophobin DEWA, lost its binding activity. DEWA_2,containing a truncated version of YaaD, maintained a moderatebinding with about 25%. Fusing the titania mineralizing peptideR553 to DEWA_2, led to a loss in the amount of the boundprotein 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. Thisis in the range of the positive control MI tooth mousse (30%).The quantitative binding results were confirmed by

fluorescence microscopy (data not shown). The proteinswhich were able to bind to hydroxyapatite in the mouthwashformulation were also tested for their binding ability to humantooth slices. For Stat_3, DEWA_2, DEWA_4, DEWA_5, andthe positive control MI tooth mousse, a qualitative binding tothe tooth slices could be visualized by fluorescence microscopy.Binding was mainly detected toward dentin, only minor bindingwas observed to enamel. The negative control, peptide #3-1,

Table 2. Results of the Binding and Nucleation Experiments

binding

HBS-T mouthwash nucleationa

amelogenin Amelo1 22 ± 5 − oAmelo2 59 ± 1 − oAmelo3 56 ± 1 − oAmelo4 60 ± 1 − oAmelo5 58 ± 5 − oAmelo6 62 ± 3 − +Amelo7 69 ± 2 − oAmelo8 60 ± 12 − oAmelo9 12 ± 2 − +

antifreezeproteins

AFP_1 33 ± 7 − +AFP_2 59 ± 13 − +AFP_3 80 ± 7 − oAFP_4 45 ± 10 − +AFP_5 60 ± 8 − +AFP_6 66 ± 6 − oAFP_7 59 ± 1 6 ± 3 oAFP_8 57 ± 2 16 ± 3 oAFP_9 91 ± 1 3 ± 2 o

hydrophobins DEWA_1 30 ± 5 − oDEWA_2 43 ± 11 25 ± 3 oDEWA_3 63 ± 1 2 ± 1 −DEWA_4 80 ± 3 36 ± 2 +DEWA_5 72 ± 1 30 ± 1 +

statherins Stat_1 75 ± 2 − −Stat_2 16 ± 4 − oStat_3 86 ± 2 14 ± 3 −Stat_4 73 ± 3 2 ± 1 −

controls MI toothmousse

70 ± 9 30 ± 1 −

(DSS)6 84 ± 9 − o#3-1 − − o

aDelayed nucleation, −; same nucleation trend, o; enhancednucleation, +.

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showed only a small amount of unspecific binding towarddentin and enamel (Figure 1).

Analysis of Nucleation Ability. To determine if thedifferent fusion constructs may enhance the formation ofhydroxyapatite, the proteins were incubated in artificial salivacontaining 1.5 mM Ca2+ and 7.3 mM PO4

3−. Mineral formationwas monitored by the detection of the decrease in c(Ca2+) inthe artificial saliva. The consumption of Ca2+ ions in thesolution is correlated with the formation of a Ca-containingmineral and is therefore an indication for nucleation. Analyzingthe time dependent deposition of mineral we observed threedifferent trends (Table 2). In the presence of no protein, themineral formation was completed after 4 h, where a stablec(Ca2+) was reached. The majority of the investigated proteinsexhibited the same nucleation behavior like the control withoutprotein.The four proteins (DEWA_3, Stat_1, Stat_3, and Stat_4)

showed a stabilizing effect, resulting in a deceleration innucleation (Supporting Information, Figure S1). The phos-phorylated statherin peptide Stat_3 decelerated the nucleation,whereas the unphosphorylated peptide (Stat_2) showed noeffect in nucleation. Also, the peptides from MI tooth mousse,one of the best binders in the mouthwash formulation,exhibited a strong stabilizing effect. Out of the nine antifreezefusion proteins, four variants (AFP_1, AFP_2, AFP_4, AFP_5)showed an increase in nucleation activity, but no bindingactivity, in the mouthwash formulation. For these proteins,almost all the Ca2+ was consumed after 3 h, as compared to 4 hfor the control without protein (Figure 2a). An even strongereffect in nucleation activity was observed with the twohydrophobin constructs DEWA_4 and DEWA_5 containingthe hydroxyapatite binding peptides, CPL1254 and P11-4,

12

respectively. After 2 h, almost no free Ca2+ could be detected inthe nucleation solution (Figure 2b).The precipitate formed in the presence of the construct

DEWA_4 or DEWA_5 and a control with no protein wascharacterized by X-ray powder diffraction. The mineral phase,including the control without protein, could be identified ascrystalline 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). Transmissionelectron microscopy of the formed mineral showed plate-likecrystals that is consistent with the morphology of hydrox-yapatite. The crystals were similar in size, but showed a

Figure 1. Binding of fluorescence labeled proteins to human toothslices in the mouthwash formulation. Bright field images of slicestreated with the positive control MI tooth mousse (a), the negativecontrol #3-1 (c), DEWA_4 (e), and DEWA_5 (g). Fluorescenceimages of the shown bright field for MI tooth mousse (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 correspondto 500 μm.

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

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

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difference in the crystal arrangement. The control samplewithout protein showed a spherical arrangement of the crystal,whereas the crystals formed in the presence of DEWA_4 andDEWA_5 showed more a bundle-like arrangement (Figure 4).

Investigation of the Crucial Amino Acids Involved inNucleation. The results of the binding and the nucleationexperiments showed that only two proteins, namely, DEWA_4and DEWA_5, have binding and nucleation activities in respectto hydroxyapatite (see Table 2). These two proteins share thesame core structure, consisting of a truncated version of YaaD,a hydroxyapatite binding peptide and a hydrophobin DEWA atthe C-terminus of the construct. They differ only in thesequence of the peptide. DEWA_4 includes the peptidesequence of CLP12 (QPYHPTIPQSVH) identified by Chunget al.54 via phage display, whereas DEWA_5 contains thesequence of P11-4 (QQRFEWEFEQQ), a rationally designedpeptide to form hydrogels.12 The construct DEWA_2(40aaYaaD-DEWA) consisting of a truncated section of theBacillus subtilis synthase YaaD and the Aspergillus nidulanshydrophobin DEWA without an inserted peptide sequenceshowed good binding to hydroxyapatite in the mouthwashformulation with about 25% bound protein. Inserting theCLP12 peptide or the P11-4 peptide enhances the bindingcapability to hydroxyapatite. Of all the investigated proteins,DEWA_4 and DEWA_5 showed the fastest decrease in calciumconcentration, whereas the construct DEWA_2 had no effecton the nucleation (see Figures 2b and 5). This suggests that thehydrophobin protein, DEWA, and the truncated YaaD inDEWA_4 and DEWA_5 are responsible for the binding to

hydroxyapatite, whereas the inserted peptides in the constructsare responsible for the nucleation activities. P11-4 by itselfshowed no effect on the nucleation (Figure 5). Beside bindingcapabilities, the fusion construct of the hydrophobin and thetruncated 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. Inorder to determine which amino acid residues play animportant role for nucleation an alanine scan was performedon the sequence of P11-4 within DEWA_5. Eight single, twodouble, and one triple mutant were constructed (see Figure6a). In addition, truncated versions of DEWA_5 were alsoconstructed. Amino acid residues of the sequence of P11-4 inDEWA_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 trendas DEWA_5 (Supporting Information, Figure S2). Also, thedouble substitution of Q49 and Q50 with alanine had no effecton the nucleation behavior. The minor impact of the glutamineresidues on the nucleation activity was further demonstrated bythe deletion variants. Deletion of the two residues Q49 andQ50, resulting in variant Δ1, as well as the additional deletionof the residues Q58 and Q59 (Δ2), had no effect on thenucleation ability of the construct. Also, variant Δ3 (40aaYaaD-FEWEFE-DEWA), containing only 6 of the originally 11 aminoacid residues of P11-4, showed the same nucleation trend as thewild-type construct DEWA_5, containing the whole P11-4peptide sequence (Supporting Information, Figure S3).The importance of the glutamate residues within the

sequence of P11-4 for the nucleation activity of the constructDEWA_5 was clearly demonstrated by the alanine and deletionvariants. Single alanine mutations at the positions E53, E55, orE57 did not influence the nucleation behavior. But the doublesubstitution of EE53/55AA resulted in a slightly slower mineralformation. The substitution of all three glutamate residues(EEE53/55/57AAA) showed a significant deceleration inmineral deposition. The deletion variant Δ4 lacking the thirdglutamate residue at position 57 exhibited a slightly delayednucleation trend as compared to the variant Δ3 or the wild-typeconstruct DEWA_5. Deletion of the second glutamate residueat position 55 (variant Δ5) led to a significantly slowernucleation (Figure 7). Since the amino acid sequence of P11-4can be reduced to six amino acids without losing nucleationactivity, the truncated variant Δ3 (40aaYaaD-FEWEFE-DEWA) was chosen to engineer the protein to a fasternucleation activity. Based on the six amino acid residuesFEWEFE within Δ3, a small focused library was designed (seeFigure 6b). Even if the alanine scan as well as the deletionvariants demonstrated the importance of the glutamate residueswithin P11-4 (QQRFEWEFEQQ), glutamate residues in variantΔ3 (40aaYaaD-FEWEFE-DEWA) were exchanged into theuncharged homologue glutamine. Furthermore, glutamate wasreplaced by aspartate, which has a shorter side chain. In orderto determine the effect of aromatic and nonaromatic aminoacids on the nucleation activity, the aromatic amino acidsphenylalanine and tryptophan in the FEWEFE-sequence werereplaced either by histidine to introduce a positively chargedresidue or by tyrosine to introduce a hydroxylated residue. Inaddition, the aromatic amino acids were exchanged to serine,arginine and valine. A single substitution of glutamate toglutamine (E50Q) caused a deceleration in nucleation (Figure

Figure 4. TEM images of the formed mineral. All samples show plate-like crystals. Crystals are spherulite arranged in the sample withoutprotein (a). In DEWA_4 (b) and DEWA_5 (c), the crystals show amore bundle-like-fibrous arrangement. Bars correspond to 200 nm.

Figure 5. Nucleation in artificial saliva. Calcium consumption in thepresence of the peptide P11-4 and DEWA_2 without an insertedpeptide.

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8). These results support that the negative charge is importantfor the nucleation activity. Similarly an exchange into aspartate(E50D) resulted in a slightly faster mineral formation. Thereplacement 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 onthe nucleation activity (Figure 8). In the case of the aromaticamino acids phenylalanine and tryptophan, a slightly highernucleation rate was observed by introducing serine and arginine(Supporting Information, Figure S4). The replacement of F49,

W51, or F53 with the aromatic homologues tyrosine andhistidine did not show any effect (Supporting Information,Figure S5). These variants had a similar nucleation behaviorlike DEWA_5. Also the substitution to valine had no effect onthe nucleation. The nonaromatic amino acid residues seem tobe more beneficial for the nucleation than the aromaticresidues.The final engineering step involved combining the beneficial

substitutions of glutamate to aspartate and of the aromaticamino acid residues phenylalanine and tryptophan to eitherserine or arginine into two new constructs named DEWA_5a(40aaYaaD-SDSDSD-DEWA) and DEWA_5b (40aaYaaD-RDRDRD-DEWA; see Figure 6b). A further increase innucleation activity in artificial saliva was observed in thepresence of these two variants as compared to DEWA_5.Nucleation reached a stable plateau after 90 min with these newvariants, whereas with DEWA_5, nucleation was completedafter 120 min (Figure 9). The analysis by X-ray powderdiffraction confirmed the formed mineral as crystallinehydroxyapatite. Major peaks were observed at 2Θ = 25.9°and 32.9° corresponding to the (002) and the (211)/(112)planes of hydroxyapatite (Figure 10). These variants were alsotested regarding their binding abilities in HBS-T buffer and the

Figure 6. (a) Schematic scheme of the construct DEWA_5, thealanine variants, and the truncated variants. The amino acid sequencesof the full length P11-4 (DEWA_5), the alanine substituted andtruncated peptides are shown between the truncated synthase YaaDand the hydrophobin DEWA protein. Alanine substitutions are boldand underlined. (b) Schematic scheme of the focused library variantsbased on the sequence of the truncated variant Δ3. Substitutions arebold and underlined, X = S, Y, R, H, V; Z = Q, D.

Figure 7. Nucleation in artificial saliva. Calcium consumption in thepresence of DEWA_5 and variants lacking glutamate residues in thesequence of P11-4 within DEWA_5 either by alanine substitution(EE53/55AA and EEE53/55/57AAA) or by deletion (Δ4 and Δ5).

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

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mouthwash formulation. For the variant EEE50/52/54DDD(40aaYaaD-FDWDFD-DEWA), a slightly higher binding wasobserved, with 76 ± 5% in HBS-T buffer and 41 ± 3% in themouthwash formulation, as compared to DEWA_5 with 71 ±6% in HBS-T buffer and 30 ± 2% in the mouthwashformulation. By introducing the serine residues, the bindingcould not be further enhanced regarding the variant DEWA_5a(73 ± 4% in HBS-T buffer, 36 ± 4% in the mouthwashformulation). But the substitution of the aromatic amino acidresidues by arginine in variant Δ3 led to a slightly higherbinding of DEWA_5b, with 82 ± 3 in HBS-T buffer and 45 ±3% in the mouthwash formulation as compared to DEWA_5(Figure 11).

■ DISCUSSIONIn order to identify new potential candidates in the field ofbiomineralization of hydroxyapatite, a panel of fusion proteinswere screened for their binding and nucleating abilities. Out ofthe nine hydroxyapatite binding proteins in the mouthwashformulation, only two fusion constructs (DEWA_4, DEWA_5)showed an increase in nucleation. That a strong binding abilitydoes not necessarily correspond to a mineralization directingactivity has been described earlier.6 It has been suggested thatproteins with low affinity toward hydroxyapatite might create a

local supersaturation by interacting with soluble ions andthereby reduce the nucleation barrier.6 This might be thereason for the low binding affinity of the antifreeze proteinsused in this study on the one hand and the increase innucleation on the other. In contrast, the peptides from MItooth mousse and Stat_3, the phosphorylated statherin peptide,revealed good binding abilities to hydroxyapatite in themouthwash formulation, but exhibited a strong stabilizingeffect on the nucleation. The peptides in MI tooth mousse arederived by trypsin digestion of the milk protein casein,55

whereas Stat_3 consists of the 21 N-terminal amino acidresidues of salivary statherin. Both proteins contain highamounts of charged residues like phosphoserine and aspartateresidues that are known to interact strongly with calcium andphosphate ion clusters.32,56 The interaction prevents furthergrowth of the ion clusters, resulting in stabilization57 andmaking milk and saliva a supersaturated solution. It is probablynot surprising that the peptides from MI tooth mousse andStat_3 revealed a strong stabilizing effect in our nucleationexperiments. In contrast, the unphosphorylated Stat_2 did notexhibit binding abilities in the mouthwash formulation andshowed no effect in the nucleation behavior. This indicates thatthe strong interaction of Stat_3, the phosphorylated peptide, isattributed to its phosphoserines.DEWA_5, the fusion construct of the truncated YaaD

protein, the mineralizing P11-4 peptide and the hydrophobinDEWA showed good binding abilities as well as an enhancednucleation in artificial saliva. The amphiphilic hydrophobinDEWA belongs to the class I of hydrophobins, which is able toform stable monolayers containing laterally arranged fibers.58

They are known to adsorb strongly to hydrophobic andhydrophilic surfaces and their high surface activity have led toapplications in surface modifications to coatings for drugnanoparticles.59−63 Recently, a fusion construct of hydrophobinHFB II and a calcium binding protein sequence has been usedfor the mineralization of calcium carbonate.64 We observedgood binding abilities to hydroxyapatite in the mouthwashformulation regarding the construct DEWA_2 (40aaYaaD-DEWA). By inserting a hydroxyapatite binding peptide thebinding could be further increased as in the case of DEWA_4(40aaYaaD-CPL12-DEWA) and DEWA_5 (40aaYaaD-P11-4-DEWA). These two constructs revealed, in addition to their

Figure 9. Nucleation in artificial saliva. Calcium consumption in thepresence of DEWA_5, the truncated variant Δ3, and the engineeredvariants EEE50/52/54DDD, DEWA_5a, DEWA_5b, and a controlwithout protein.

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

Figure 11. Quantitative binding results of DEWA_5 and the variantsEEE50/52/54DDD, DEWA_5a and DEWA_5b to hydroxyapatite inHBS-T buffer (white bars) and the mouthwash formulation (graybars).

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binding activity, an enhanced nucleation activity. NeitherDEWA_2 nor only the peptide P11-4 showed an effect innucleation, whereas the fusion construct DEWA_5 exhibits astrong increase in mineral formation. P11-4 is a rationallydesigned peptide. Above a concentration of 15 mg/mL, thispeptide is able to self-assemble into a fibrillar network servingas a template for nucleation.12 In our experiments, theconcentration of the peptide P11-4 was much lower (25 μMcorresponds to a concentration of 0.04 mg/mL) and no self-assembly of the peptide was observed, which might be thereason why no increase in nucleation was observed. Thisdemonstrates the importance of having a rigid scaffold toenhance nucleation. Many proteins exhibit a mineralizingactivity after immobilization on a solid scaffold or after self-assembly.8−11,65−67 In solution, these proteins failed tomineralize hydroxyapatite. In our work, the stabilization ofP11-4 in the construct DEWA_5 was not observed by self-assembly of the peptide, but achieved by fusion to DEWA andYaaD. Beside binding abilities, this fusion protein might also beacting as a scaffold.The peptide P11-4 was rationally designed to self-assemble.

The self-assembly process is triggered in a pH-dependentmanner by the glutamic acid residues. The terminal glutamineresidues contribute to self-assembly by increasing the numberof hydrogen bonds.12,68 Therefore, it is perhaps not surprisingthat deletion of the terminal glutamine residues does not affectthe nucleation behavior as they are only introduced into thepeptide sequence to trigger the self-assembly. That acidicamino acid residues (E53, E55, E57) play a key role inbiomineralization processes has been widely described.9,65,69,70

The alanine scan and also the deletion variants of DEWA_5support this. By changing the glutamate residues into alanine ordeleting these residues, a decrease in nucleation activity wasobserved. On the other hand, the substitution of the glutamateresidues within the truncated variant Δ3 by aspartate led tohigher binding ability. This is consistent with other experimentsfound in literature that show that aspartate residues have ahigher binding affinity toward hydroxyapatite then glutamateresidues.9,69,71,72 Additionally, the fusion construct DEWA_5could be further engineered toward a faster mineral formation.The engineered variant DEWA_5a contains the sequence

(SD)3. By comparing this sequence to proteins that arenaturally involved in the formation of hydroxyapatite thesimilarity to the motif D-S-S in the dentin phosphoprotein isobvious. Beside the predominate motif D-S-S, also repeats of(DS) were found in the sequence of DPP.25,73 The furtheroptimization of the nucleation activity of the original peptideP11-4 embedded in the 40aa Yaad-DEWA construct(DEWA_5) led to an amino acid motif that evolved in thebiomineralization of hydroxyapatite. It is noteworthy to pointout that the serin residues in the wildtype dentinphosphoprotein are phosphorylated, whereas the serin residuesin DEWA_5a are unphosphorylated. Although phosphorylationplays an important role in vivo biomineralization ofhydroxyapatite by this motif, Yarbrough and co-workers couldshow that the unphosphorylated (DSS)n-motif inherits also anucleation activity when being conformational stabilized. Thiswas shown by the immobilization of (DSS)8 onto polystyrenebeads.9 The engineered motif (SD)3 is embedded in the 40aaYaaD-DEWA construct and thereby in a conformational lessflexible state than as the peptide alone in solution. Inserting thepredominate motif (DSS)n into the 40aa Yaad-DEWAconstruct (DEWA_2) would be interesting, as we already

observed a higher nucleation rate with the motif (SD)3. Theoriginal peptide P11-4 was designed to mimic proteins ofextracellular matrices like in nature.12 Within the fusion protein(DEWA_5), this sequence was subsequently modified into anamino acid motif (SD)3 that is found in nature.

■ CONCLUSIONSThe work described herein has several significant implicationsin protein/peptide-assisted biomineralization. First, the studyhas identified fusion proteins that are able to bind to andnucleate hydroxyapatite. Strong binding affinity to hydrox-yapatite is not necessarily an indication of its nucleation activity.Second, certain amino acid residues were found to play a role inthe binding ability and nucleation of hydroxyapatite. We coulddemonstrate that nonaromatic, hydroxylated (S), and charged(R/D) amino acid residues show beneficial effects in thebinding and nucleation of hydroxyapatite. The sequenceresponsible 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), wehave engineered a unique hydroxyapatite-binding motif (SD)3with biological nucleation activity within a fusion protein.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.bio-mac.6b00135.

The data for the nucleation of the statherin variants,alanine scan single variants, and variants lackingglutamine residues in the sequence of P11-4 withinDEWA_5 (PDF).

■ AUTHOR INFORMATIONCorresponding Author*Phone: 0049-711-685-63193. Fax: 0049-711-685-64569. E-mail: bernhard.hauer@itb.uni-stuttgart.de.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe would like to acknowledge BASF SE, Ludwigshafen,Germany, for their financial support. We especially thank Dr.Nina Schneider, Dr. Stefan Jenewein, and Dr. ClausBollschweiler for many helpful discussions. We are grateful toMoritz Schmelz, Institute of Mineralogy, University ofStuttgart, for the preparation of the tooth slices. We alsothank Dr. Thomas Theye, Institute of Mineralogy, University ofStuttgart, for the XRD measurements and Dr. StephanNußberger and Dr. Michael Schweikert of the Institute ofBiology, University Stuttgart, for the TEM measurements.

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Biomacromolecules Article

DOI: 10.1021/acs.biomac.6b00135Biomacromolecules XXXX, XXX, XXX−XXX

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