The STNA15-ELP was acquired from Technical Proteins Nanobiotechnology (Valladolid, Spain). These recombinant proteins are produced by genetically modified bacteria and extracted at the company (Girotti et al., 2004). The full sequence of the protein used in this study is MESLLP-[((VPGIG)₂VPGKG(VPGIG)₂)₂-DDDEEKFLRRIGRFG-((VPGIG)₂VPGKG(VPGIG)₂)₂]₃-V where the STNA15 sequence is highlighted in bold. It has an inverse transition temperature (ITT) of 23°C and isoelectric point (pI) at pH 9.9 (details provided by the supplier).

To prepare the stock protein solution, 1 mg of STNA15-ELP was dissolved in 1 ml of ultrapure water (18 MΩ·cm). The stock solution was then diluted to yield a 100 μg/ml concentration. To prepare constrained protein samples, 100 μl of the dilute solution was pipetted onto borosilicate glass slides (VWR International Ltd, Lutterworth, UK) and left to completely dry at 21°C overnight.

To prepare the unconstrained STNA15-ELP samples, 100 μl of the 1 mg/ml protein stock solution was pipetted directly into 1 ml of the mineralizing solution. After the mineralization period of either 3 h or 8 days, the samples were frozen in liquid nitrogen and lyophilized. The lyophilized samples were washed with ethanol and dried prior to chemical and morphological analysis.

The mineralizing solution was used as previously described by Chen et al. (2006). In short, 104.7 mg sinter grade HAp (Plasma Biotal Ltd., Derbyshire, UK) and 8.49 mg sodium fluoride (Sigma Aldrich, UK) was added to 100 ml of ultrapure water (18 MΩ·cm). Sixty nine percent analytical grade nitric acid (VWR International Ltd., Lutterworth, UK) was added drop-wise until the solution became clear and colorless and a pH of 2.4 was reached. Twenty eight to thirty percent ammonium hydroxide (Sigma Aldrich, UK) was added drop-wise to the solution until pH of 6 was reached. The solution was prepared at room temperature and stirred continuously during preparation.

Three milliliters of solution were incubated with each coated sample and 1 ml with STNA15-ELP solution samples. For the coated samples, uncoated borosilicate glass was incubated in the mineralizing solution as a control. Precipitate from pure mineralizing solution, without any protein, was used as a control for unconstrained protein samples. All samples were incubated in the mineralizing solution for either 3 h or 8 days at 37°C in sealed containers.

Quartz crystal microbalance measurements were carried out in order to check the affinity of the protein to the borosilicate glass. Borosilicate coated quartz sensor crystals (Biolin Scientific Ltd, Stockholm, Sweden) were first washed in a 2% w/v SDS (Sigma Aldrich, UK) solution for 30 min followed by a 10 min UV/ ozone treatment as a cleaning procedure. In the QCM (Q-sense, Biolin Scientific Ltd, Stockholm, Sweden) the crystal was stabilized in ultrapure water then, a solution of STNA15-ELP (100 μg/ml) was added and the QCM measurement was taken until equilibrium was reached. Finally, the crystal was washed again with ultrapure water to remove any unbound or loosely bound protein. The change in resonant frequency of the quartz crystal was converted to mass of protein adsorbed using the Sauerbery equation (Equation 1) where Δm is the change in mass, Δf the measured change in frequency upon protein adsorption, n is the overtone number (3) and C is a constant specific to the crystal.

A DSA100 Drop Shape Analyzer from Krüss (Hamburg, Germany) was used to measure the contact angle of water on uncoated and coated borosilicate glass slides. Five microliters of water were pipetted onto the slide and the angle measured immediately. Three repeats were taken and averaged.

Scanning electron microscopy (SEM) images were recorded, using a secondary electron (SE) and back-scattered electron (BSE) detectors, on an FEI Inspect F SEM (Hillsboro, Oregon, USA) in the NanoVision Centre, Queen Mary University of London. An X-Act Oxford Instruments EDX detector was used for EDX measurements (20 kV accelerating voltage) (Abington, Oxfordshire, UK). The samples were coated with carbon for EDX studies and 20 nm of gold for morphological examination.

TEM images and SAED patterns were recorded on a JEOL 2010 transmission electron microscope operated at 200 kV (Tokyo, Japan). For the 3 h incubation timepoint of constrained ELP mineralization, the crystals were grown directly on a TEM grid. The 8 day mineralized samples were scraped off the borosilicate glass into ethanol and then pipetted onto grids.

The 3 h and 8 day unconstrained samples were first suspended in ethanol then pipetted onto copper grids and dried prior to TEM inspection.

ATR-FTIR was carried out using a Bruker Tensor 27 IR spectrometer (Billerica, Massachusetts, USA) to analyze both protein conformation and chemical groups of the mineral precipitate. STNA15-ELP was prepared as previously described with the ultrapure water exchanged for deuterium oxide (D₂O) (VWR International Ltd, Lutterworth, UK).

Before FTIR measurement, an STNA15-ELP coated glass slip was rinsed with D₂O to remove excess protein. The measurements on the coating were taken in a wet condition. Free protein samples were pipetted directly on the ATR window. Eighty scans per measurement, 400–2,000 cm⁻¹ range, 3 repeats were acquired and averaged. The FTIR chamber was purged with nitrogen during readings. The amide I region was deconvoluted in Origin Pro using the Gaussian fit and previously reported literature values (Serrano et al., 2007). For the characterization of the mineral precipitate, 60 scans, in the range of 400–4,000 cm⁻¹, were taken for each mineralized sample. All measurements were carried out at 21°C.

Since the FTIR spectrum of borosilicate glass has large absorptions in the range where calcium phosphate peaks are present, Raman spectroscopy was chosen to analyze the mineral grown on constrained ELP surfaces. A Renishaw inVia Raman Microscope (Wotton-under-Edge, Gloucestershire, UK), equipped with a 633 nm wavelength (20 mW power) laser, was used to record the spectra of the mineral formed in the presence of constrained STNA15-ELP at 3 h and 8 days. A 20x objective was used, giving a spot size of 1.93 μm. The spectra were obtained in the 170–1370 cm⁻¹ range with an exposure time of 5 s. Sixty accumulations were recorded for each reading.

The contact angle of the borosilicate glass surface displayed in Table 1 is indicative of a surface with low hydrophilicity (θ = 70.9°C). Reports have shown that protein adsorption is favored when θ > 65°C (Vogler, 2012). Under the given experimental conditions, according to literature value for the isoelectric point (pI), the glass carries a net negative charge (Blass et al., 2013). The protein can arrange itself on the glass surface in such a way that the negative STNA15 (mineralizing) sequence is exposed to the solution due to repulsion from the negatively charged glass surface. The positively charged lysine residues in STNA15-ELP are expected to interact with the negative glass surface. The QCM study confirmed that the STNA15-ELP, in ultrapure water, does bind to the borosilicate glass. The study showed a good binding of STNA15-ELP where 15.2 mg/m² remained adsorbed to the substrate after washing with ultrapure water (Figure 1B). The contact angle of the coating suggests that the STNA15-ELP coating formed a hydrophilic surface when adsorbed to the glass (θ = 18.7°C, Table 1), suggesting the exposure of the hydrophilic mineralizing sequence on the glass surface.

The FTIR spectra of STNA15-ELP in solution vs. STNA15-ELP coating are shown in Figure 2. There is a noticeable difference in the amide I (1,600–1,700 cm⁻¹) and amide II (1,400–1,500 cm⁻¹) relative intensities (Figure 2A). The amide peaks are normally made up of several components arising due to the stretching vibration of C = O (amide I) and C-N and N-H bending (amide II) (Lenk et al., 1991). The position of the peaks changes depending on the secondary structure of the protein as the hydrogen bonding within the protein alters upon adsorption and/or conformational changes. The dipole moments of the amide I and amide II peaks are almost perpendicular to one another (Miyazawa and Blout, 1961), hence the change in amide I and amide II intensity is consistent with differences in conformation between the bound and unbound proteins. The overall amide I peak increased in intensity when STNA15-ELP was adsorbed on glass, conversely there was a reduction in the amide II intensity, compared to STNA15-ELP prior to adsorption (Figure 2A). Conformational changes of the ELP, upon binding, agree with previous reports of natural elastin structural changes during surface adsorption. Natural elastin is globular in solution and undergoes conformational changes in order to assemble into an ordered structure upon surface adsorption (Subburaman et al., 2006).

To investigate further, the solution (Figure 2B) and coated (Figure 2C) samples' amide I peaks were deconvoluted using a Gaussian fit. The peak values were chosen according to previous indexed values in literature (Serrano et al., 2007). The results indicate a relative increase in the amount of β-sheet/β-turn structure (at 1,647 cm⁻¹), when the protein was constrained on glass, accompanied with a relative decrease in the random coil component (at 1,660/1,661 cm⁻¹) (Table 2). The increase in the amount of β-sheet/β-turn structure and a decrease of the random coil in the amide I band is indicative of an increase in conformation of the secondary structure of STNA15-ELP when adsorbed on a glass substrate.

ELPs are known to have thermoresponsive properties, attributed to their ITT (Urry et al., 1998). It is known that below the ITT the protein is unfolded, mostly with a random coil structure, and the hydrophobic residues are hydrated with clathrate-like water structures (Rodriguez-Cabello, 2004). Above this temperature, the ordered water molecules surrounding the protein are disrupted and the protein becomes dehydrated. The dehydration process causes the protein to fold, via an increase in the β-sheet/β-turn component and simultaneous decrease in random coil, and phase separate. This process is not instantaneous and structural changes occur over a range of temperatures above and below the ITT (for example Reiersen et al., 1998). Previous work by Serrano et al. (2007) investigated ELP structures with FTIR and concluded that below the ITT the protein contains the β-sheet aggregation component (1,616 cm⁻¹), but this completely disappears upon heating above the ITT. The FTIR data presented here shows a similar change between the unconstrained and the constrained STNA15-ELP. From the results presented in Figure 2 and Table 2, it can be expected that upon adsorption on the glass surface the ELP folds into a similar conformation as it would when heated well above the ITT, i.e., the protein loses the random coil component and gains order in its secondary structure.

Protein adsorption is a complex process and can change dramatically with alteration in the surface and solution chemistry. A number of factors that affect adsorption of proteins onto surfaces have been identified. These include pH (pH close to pI promotes adsorption) (Norde, 1986; Norde et al., 1986); substrate hydrophobicity (Shirahama and Suzawa, 1985); dehydration (Lee and Ruckenstein, 1988); and surface-solution equilibrium (Wojciechowski et al., 1986). Although protein adsorption mechanisms can be the result of multiple factors, the adsorption behavior is specific to each protein and the surface it is adsorbing to. In this study, the STNA15-ELP is dissolved in ultrapure water (pH ~7.0) and is far from its pI. Thus, according to Norde et al. (1986), it would not favorably adsorb to the glass. However, the QCM results (Figure 1) confirm that the protein is well bound to the glass surface. Lee and Ruckenstein (1988) proposed that proteins gain entropy upon adsorption to surfaces due to dehydration and structural changes. This explanation is more suitable for the system described here, since STNA15-ELP appears to gain conformation upon adsorption and this is known to occur during the dehydration of the ELP chains and subsequent hydrophobic collapse.

Differences were observed in the mineralization process and the FAp morphology obtained at the 8 day incubation period between the constrained and unconstrained STNA15-ELP mineralized samples. In the case of the constrained protein, the ordered STNA15-ELP on the glass surface appeared to restrict the formation of the FAp to polycrystalline platelet morphology. The results strongly suggest that there are two reasons for this. Firstly, the brushite platelets exist for longer periods of time in the presence of STNA15-ELP coating. Reports have shown that other proteins, such as bovine serum albumin, can retard the transformation of brushite to apatite (Xie et al., 2001, 2002). Although there is no clear explanation for this, it is speculated that the adsorbed protein prevents water molecules from making direct contact with the surface of the brushite and therefore prevents dissolution.

The second explanation is based on templated mineralization. The stabilized brushite crystals, surrounded by the protein, slowly dissolved once formed. The dissolution of the crystals was evidenced by the etch pits visible on the surface (Figure 3B). As brushite formed, along with the calcium fluoride precipitate, the calcium-pH isotherm shifts to an area where brushite was no longer stable, causing dissolution. The brushite dissolution created a local supersaturation of calcium that could reprecipitate as FAp, in the presence of fluoride. The slow FAp formation process allowed an ordered structure to form where the FAp crystallites grow along the {010} face of brushite.

A different process occurred when the protein was unconstrained and free in solution. As seen in Figure 3B, the nucleation was random and the growth of the crystals had no preferred orientation. The protein aggregates created an environment which favored apatite formation over other phases, even at 3 h of incubation. This may be due to local buffering effects caused by the lysine amino acids in the STNA15-ELP sequence. Since the lysine groups are likely to interact with the borosilicate surface, this effect is not seen with the STNA15-ELP coating. Other mineralizing organic molecules, such as amelogenin, have shown capability of buffering pH and stabilizing precursor phases. Amelogenin has the ability to stabilize amorphous calcium phosphate in a solution which normally precipitates apatite (Kwak et al., 2011).

The specific behavior observed in this study can be further supported by the thermoresponsive properties of ELPs related to their ITT. ELPs are known to fold and aggregate above their transition temperature. In its folded state, the STNA15-ELP can display, when in solution, its nucleating sites on the surface causing FAp nucleation all over the aggregate. The nucleation on ELP aggregates is consistent with other work where needle like apatite grew from spherical ELP particles (Misbah et al., 2016). However, once adsorbed on glass and with an increase in order, the STNA15 sequence of the ELP is no longer concentrated on a surface of a particle but on a flat substrate. In both cases the mineral formation resembled the process of biomineralization where either a precursor phase was present before a stable mineral is formed (for example Johnsson and Nancollas, 1992) or smaller sub-units fused to form larger, imperfect crystals (for example Robinson, 2007). However, only the constrained protein produced mineral that has a preferential growth direction.

These findings provide information that can lead to synthesis of ordered FAp structures that could be used in enamel therapeutics. However, more importantly, this synthetic biomineralizing system has shown that protein constraint and protein conformation both play an extremely important role in the process of biomineralization. Protein constraint can be related to natural biomineralizing processes, where the mineralizing proteins exist within a gel-like matrix. In fact proteins, such as amelogenin, have been shown to assemble into fibers and nanospheres, rather than existing in a free form (Carneiro et al., 2016).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer YZ and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.