Changes in the Secondary Structure and Assembly of Proteins on Fluoride Ceramic (CeF3) Nanoparticle Surfaces

Fluoride nanoparticles (NPs) are materials utilized in the biomedical field for applications including imaging of the brain. Their interactions with biological systems and molecules are being investigated, but the mechanism underlying these interactions remains unclear. We focused on possible changes in the secondary structure and aggregation state of proteins on the surface of NPs and investigated the principle underlying the changes using the amyloid β peptide (Aβ16–20) based on infrared spectrometry. CeF3 NPs (diameter 80 nm) were synthesized via thermal decomposition. Infrared spectrometry showed that the presence of CeF3 NPs promotes the formation of the β-sheet structure of Aβ16–20. This phenomenon was attributed to the hydrophobic interaction between NPs and Aβ peptides in aqueous environments, which causes the Aβ peptides to approach each other on the NP surface and form ordered hydrogen bonds. Because of the coexisting salts on the secondary structure and assembly of Aβ peptides, the formation of the β-sheet structure of Aβ peptides on the NP surface was suppressed in the presence of NH4+ and NO3– ions, suggesting the possibility that Aβ peptides were adsorbed and bound to the NP surface. The formation of the β-sheet structure of Aβ peptides was promoted in the presence of NH4+, whereas it was suppressed in the presence of NO3– because of the electrostatic interaction between the lysine residue of the Aβ peptide and the ions. Our findings will contribute to comparative studies on the effect of different NPs with different physicochemical properties on the molecular state of proteins.


■ INTRODUCTION
In the field of material sciences, the self-assembly of molecules is a major area of interest for researchers to develop soft matter. 1 Furthermore, in biology, the secondary structure and assembly state of a protein are important for regulating the function and activity of the protein. The formation of fibrillar aggregates of peptides and proteins is associated with various diseases including neurogenerative disorders. 2 The dysregulation of the structure and state of proteins can lead to diseases in biological organs. 3,4 For example, fibril formation via the self-assembly of denatured proteins causes amyloid diseases, such as Alzheimer's disease, 5 which is the most common neurodegenerative disease and is associated with cognitive and physical decline. 6 Pathologically, in patients with Alzheimer's diseases, senile (neuritic) plaques of the amyloid beta (Aβ) protein are observed in the brain tissue. 7 Amino acid residues in proteins interact with each other through hydrogen bonds, disulfide bonds, electrostatic interactions, and hydrophobic interactions; these interactions affect protein conformation. As the distance between peptides reduces due to protein aggregation, their secondary structure can change due to an increase in interactions among peptides, for example, an increase in the β-sheet structure via enhanced hydrogen bonds. Although Aβ generally aggregates more at higher concentrations, even at low concentrations, it forms a βsheet structure that can promote self-assembly via aromatic interactions between phenylalanine residues. 8,9 Especially, the hydrophobic domain of Aβthe region around residues 17− 20, LVFFis important for β-sheet formation. 10 In general, changes in the secondary structure and protein aggregation can be enhanced at liquid−liquid and liquid−air interfaces, as observed through denaturation by surfactants. In addition to these interfaces, liquid−solid interfaces are also likely to be the sites for protein denaturation because the domains with high affinity for the solid are exposed to the molecular surface. Molecular dynamics (MD) simulations have suggested that the structural arrangement of Aβ attached to the surface of carbon nanotubesan inorganic nanomaterial changes with the radius of the nanotubes. 11 The rate of peptide aggregation on the solid−liquid interface is determined by the affinity (binding force) between the peptide and the solid material and the shape (roughness) of the solid surface. 12 While a high affinity between peptides and solid surfaces enhances their adsorption and inhibits the self-assembly (aggregation) of peptides, solid materials with middle affinities and a rough surface (with a nanoscale morphology) accelerate the aggregation of peptides. In contrast, in a study in which allatom MD simulations of the conformation of Aβ 16−22 peptides were performed, hydrophobic interactions were found to prevent the formation of β-sheet structures in the presence of gold nanoparticles (NPs). 13 The charge on the surface of NPs is also likely to be important for Aβ fibrillation. Negatively charged NPs inhibit the formation of Aβ fibrillation, whereas positively charged NPs have no effect on fibril formation. 14 The aggregation dynamics of Aβ peptides (Aβ 16−21 ) on fullerene NP models was also investigated using MD simulation along with the effect of the coexistence of ionic salts. 15 However, there has still been no experimental evidence of how Aβ peptides behave on NP surfaces in physiological environments containing salts.  In the present study, fluoride ceramic NPs, which are expected to be applied to the biomedical field, were used as a target material. Fluoride has a moderate phonon energy of 350 cm −1 and has been widely utilized in the biomedical field for applications such as brain imaging 16−18 as a fluorescent contrast agent containing rare-earth ions. 19−22 This is because it has both high chemical durability, as a lower phonon energy reduces the chemical stability similarly to chlorides, and high luminescence efficiency when doped with rare earths because higher phonon energy causes quenching via enhanced thermal relaxation. 23 Fluoride nanomaterials labeled within the longwavelength (>1000 nm) near-infrared (NIR) region, called the second and third NIR (NIR-II/III) biological windows, 24 have been developed for NIR fluorescence computed tomography, 25 photodynamic therapy, 26 and fluorescence nanothermometers 27,28 for in vivo investigations of deep tissues such as tissues in the peritoneal cavity. 29 Rare-earth-doped fluoride crystals have also been developed for application in lifetime-based NIR fluorescence thermometers. 30 Fluoride crystals containing Gd 3+ (e.g., NaGdF 4 ) and luminescent rare earths have been developed for bimodal imaging in fluorescence and magnetic resonance modalities. 31−36 CeF 3 NPs were used in this study as a fluoride ceramic that can show a surface reactivity similar to that of the fluorides, as mentioned above. The aim of this study was to investigate the effect of fluoride ceramic NPs, which are expected to have further biomedical applications, on the conformation and assembly of Aβ molecules using an in vitro experimental system and MD simulation.

■ RESULTS AND DISCUSSION
Fluoride NPs synthesized in this study were characterized using X-ray diffraction (XRD) and dynamic light scattering (DLS). As shown in Figure 1a, the XRD patterns showed that the NPs were majorly CeF 3 with small amounts of NaCeF 4 . Data from DLS showed that the CeF 3 NPs showed a major peak at a diameter of 80 nm with a low polydispersity index (0.116), although it contained a minor fraction peak at 20 nm. We considered the representative CeF 3 particle size to be 80 nm of the peak in the size distribution in our following investigations.
In this study, fragment peptides with small molecular weights, Aβ 16−20 , which allow MD simulations to be performed easily, were used to investigate the secondary conformational changes and aggregation of Aβ using both Fourier transform infrared (FT-IR) spectroscopy and the MD simulations. CeF 3 NPs were dispersed in an aqueous solution to interact with Aβ 16−20 (KLVFF) in the aqueous solution. FT-IR spectroscopy was used for analyzing the secondary structure of proteins. Especially, the amide I vibration peak (appearing around 1650 cm −1 and mainly attributed to CO stretching) was focused because it is hardly affected by the nature of the side chains but depends on the secondary structure of the backbone. 37 Thus, it is commonly used for the secondary structure analysis 38 that can also be applied to in situ analyses under microscopy. 39 Not only the secondary structure but also the aggregation of the Aβ peptide in solvents can be analyzed using FT-IR spectroscopy. 40 Hydrochloric acid (1 mmol/L) was used to maintain the dispersibility of the CeF 3 NPs. However, the water molecule showed peaks not only at 3300 cm −1 but also at 1650 cm −1 in the IR region; these peaks interfere with those of the amide I band at 1650 cm −1 , 9 which is the target of analysis in this study. Therefore, deuterium chloride and deuterium oxide were used, instead of hydrochloric acid and water, as the dispersion media for CeF 3 and solution of Aβ 16−20 . Deuterium chloride did not affect the FT-IR spectra of Aβ 16−20 solution at this concentration (final 0.25 mmol/L) (data not shown). FT-IR spectra of the samples in which Aβ 16−20 was interacted with different concentrations of CeF 3 NPs were analyzed. As shown in Figure 2a, Aβ 16−20 showed two major peaks at 1674 and 1640 cm −1 , which correspond to aggregates and monomers, respectively. 9 Deconvolution analysis using Gaussian fitting showed that the FT-IR absorption spectra of Aβ 16−20 also included, in addition to the major peaks, a minor peak at 1618 cm −1 corresponding to β-sheet formation of Aβ 16−20 9,38 ( Figure 2b). The β-sheet formation of Aβ 16−20 (6 mg/mL) increased in the presence of 3 mg/mL CeF 3 NPs, and this increase was not observed in the presence of 6 mg/mL CeF 3 NPs as the ratios of the β-sheet peak in the total amide I absorption were 6.8, 9.3, 6.9, and 6.3% in Aβ 16−20 that interacted with 0, 3, 6, and 9 mg/mL of CeF 3 NPs, respectively (Figure 2b−e and Table 1). This may be due to the difference in the number of Aβ molecules per surface area of the NPs, which is the site of the NP−protein interaction in the system. Because the shape of CeF 3 NPs (density: 6.16 g/cm 3 ) is approximated to be a sphere with a diameter of 80 nm (2.7 × 10 5 nm 3 /particle), the mass and surface area are 1.7 × 10 −15 g and 2.0 × 10 4 nm, 2 respectively, leading to a specific surface area per mass of 1.2 × 10 19 nm 2 /g. The surface area of the CeF 3 particles in a dispersion of 3 mg/mL was 3.7 × 10 16 nm 2 / mL, while the total particle surface area in the dispersion was proportional to the particle concentration. Although the ratio of Aβ molecules that were attracted to the NP surface, that is, their local enrichment rate on the surface, in the dispersion was unknown, our results suggest that a certain enrichment of Aβ molecules promotes intermolecular bonding, thereby promoting β-sheet formation.
The effect of coexisting ions on the behavior of the Aβ 16−20 peptide on the surface of CeF 3 NPs was studied using Aβ 16−20 in D 2 O with dissolved NaCl, NH 4 Cl, and NaNO 3 (0.15 M). The effects of these ions at the same concentration were investigated in this experiment to compare the principle of action of each ion. Even without CeF 3 NPs, NH 4 + promoted βsheet formation of Aβ 16−20 , whereas NO 3 − enhanced the monomer retention of Aβ 16−20 (Table 2). Na + and Cl − did not   (Figure 3). The results suggest that NH 4 + and NO 3 − suppressed the β-sheet formation of Aβ promoted on CeF 3 . MD simulations were further performed on four monomers of Aβ 16−20 in the presence of these salts. The findings showed that the NO 3 − was strongly bound to the peptide as compared to chloride in the absence of NPs. The average distance between the peptide and NO 3 − was 0.67 nm, whereas the distance with Cl − was 0.99 nm (Table 3). Elevated peaks were observed on the radial distribution function (rdf) plots between peptide residues and NO 3 − and Na + of NaNO 3 with maximum peak values of ∼9.5 at a 0.42 nm distance, as shown in Figure 4a, and ∼0.88 at a 0.97 nm distance, as shown in Figure 4b). In contrast, comparatively low peak values were observed between peptides and ions of NaCl and NH 4 Cl (with maximum peak values of ∼0.96 at a 0.95 nm distance, as shown in Figure 4a, and ∼0.38 at a 1.0 nm distance, as shown in Figure 4b), indicating their weak interactions. Among the different residues of Aβ 16−20 (KLVFF), NO 3 − strongly interacted with the lysine (K-16) residue, with the average distance between lysine and NO 3 − being 0.4 nm ( Table 3). The possible reasons for lysine and NO 3 − interactions are as   follows: 1) the strong electrostatic interactions between positively charged lysine and anions and 2) the formation of hydrogen bonds between lysine's sidechain and NO 3 − . 41 Consequently, these strong interactions suppressed the formation of β-sheets in the secondary structures of Aβ 16−20 peptides in the NaNO 3 environment. Representative snapshots of the systems under study are shown in Figure 5. In addition to NaNO 3 , as shown in Figure 4b and Table 3, the cation of NH 4 Cl interacted with the phenylalanine (F) residues of Aβ 16−20 via cation−π interactions, which might have enhanced the β-sheet formation. 42 The solvent accessible surface areas (SASAs) of the peptides were further studied to compare the aggregation kinetics under different environments ( Figure 6). The total SASA (SASA 0 ) of the four peptides in the beginning of the simulation was 39 nm 2 , which during the 100 ns of the simulations, decreased to ∼22 nm 2 (SASA 100 ), indicating peptide aggregation ( Figure  6a). The initial aggregation kinetics was quantified by estimating the time when the total SASA of peptides reached 34 nm 2 (SASA 34 ) during the first 30 ns of the simulations (Figure 6b). According to SASA plots (Figure 6b), enhanced aggregation kinetics was observed in the presence of 0.15 M NH 4 Cl (SASA 34 was reached in 8 ns), which was related to the enhanced formation of beta sheets, observed from IR spectra (as shown previously in Table 2). The slowest aggregation kinetics was observed in the system with 0.15 M NaNO 3 (SASA 34 was reached in 19 ns), which corresponded to the retention of monomers in this environment, consistent with the results of IR absorption (shown previously on Table 2).

■ CONCLUSIONS
We evaluated the changes in the secondary structure and assembly of the Aβ peptide (Aβ 16−20 ) due to CeF 3 NPs using liquid film FT-IR measurements and MD simulations. CeF 3 NPs were found to locally concentrate Aβ 16−20 on their surfaces, possibly due to the hydrophobic interaction between NPs and Aβ 16−20 in aqueous environments, and promote the βsheet formation of Aβ 16−20 . The concentrated Aβ 16−20 on the NP surface formed ordered hydrogen bonds to form a β-sheet. This increase in the β-sheet formation of Aβ 16−20 on the NP surfaces was suppressed in the presence of NH 4 + and NO 3 − ions. Hydrogen bonding between Aβ peptides were dominant when concentrated on CeF 3 NP surfaces in the absence of NH 4 + or NO 3 − . In the presence of NH 4 + or NO 3 − , the hydrogen bonding was suppressed due to dominant bonding between the NPs and Aβ peptides. The formation of the βsheet structure of Aβ peptides was promoted in the presence of NH 4 + ions, whereas it was suppressed in the presence of NO 3 − ions regardless of the presence/absence of CeF 3 NPs, which can be explained by the electrostatic interaction between the lysine residue (amino group) of Aβ peptides and the ions. Although this study was performed using the Aβ 16−20 peptide, future research will be conducted using full-length Aβ (Aβ 1−42 ) to reveal more realistic in vivo phenomena. The analysis technique using FT-IR spectroscopy and MD will contribute to comparative studies of the effect of NPs on the molecular state of proteins under various physicochemical conditions.
FT-IR Spectroscopy for Samples in Solution. Fluoride NPs (27 mg/mL) in cyclohexane (750 μL) were slowly added dropwise into 1 mmol/L DCl solution in D 2 O (750 μL) and stirred for 16 h to remove cyclohexane via evaporation and to exchange the dispersion media with DCl/D 2 O. Aβ 16−20 was dissolved in D 2 O at 8 mg/mL. The Aβ 16−20 solution (8 mg/mL) in D 2 O with and without NH 4 Cl, NaNO 3 , or NaCl (0.2 M) was mixed with different concentrations (3−27 mg/mL) of the NP dispersion in 1 mmol/L DCl solution at a 3:1 volume ratio (thus, the final concentration of Aβ 16−20 in the mixed samples was 6 mg/mL). The final concentrations of the NPs were set at 1−9 mg/mL because the concentration order of milligrams per milliliter is the dose commonly used for imaging contrast agents for visualizing blood flow. 27,36,44 FT-IR spectra including amide bands were recorded using an FT/IR-6200 spectrometer (Shimadzu Co., Kyoto, Japan) for the mixed samples sandwiched between two CaF 2 plate windows (spacer 0.025 mm). The analysis was performed for each sample within 30 min after mixing Aβ 16−20 with the NP dispersion.
MD Simulations. MD simulations were performed using GROMACS 2019.6 software with a GROMOS 54A7 force field. Four Aβ 16−20 peptide monomers with a concentration of 6 mg/mL were inserted in a 9 × 9 × 9 nm 3 box. The simulations were performed in the absence of salts and in the presence of 0.15 M NaCl, NH 4 Cl, and NaNO 3 solutions. The MD run was performed for 100 ns for each system, following the methodology described in a previous study. 15 Analysis of the MD Simulations. Formation of peptide aggregates and kinetics of aggregation were studied via SASA analysis. The interactions between ions and peptide residues were studied in the last 10 ns of the simulations, when the peptide aggregates were produced. The rdf and intermolecular distance analyses were performed using the centers of mass of the peptides, averaged among four peptides. Visual molecular dynamics (VMD) software was used for the visualization of the systems under the study.