Aluminium Binding to Modified Amyloid-β Peptides: Implications for Alzheimer’s Disease

Aluminium (Al) is clearly neurotoxic and considerable evidence exists that Al may play a role in the aetiology or pathogenesis of Alzheimer’s disease (AD). Nevertheless, the link between AD pathology and Al is still open to debate. Therefore, we investigated here the interaction of aluminium ions with two Aβ peptide fragments and their analogues. First, we synthesised by the Fmoc/tBu solid-phase peptide synthesis (SPPS) strategy using an automated peptide synthesiser two new peptides starting from the Aβ(1–16) native peptide fragment. For this purpose, the three histidine residues (H6, H13, and H14) of the Aβ(1–16) peptide were replaced by three alanine and three serine residues to form the modified peptides Aβ(1–16)A36,13,14 and Aβ(1–16)S36,13,14 (primary structures: H-1DAEFRADSGYEVAAQK16-NH2 and H-1DAEFRSDSGYEVSSQK16-NH2). In addition, the Aβ(9–16) peptide fragment (H-9GYEVHHQK16-NH2) and its glycine analogues, namely Aβ(9–16)G110, (H-9GGEVHHQK16-NH2), Aβ(9–16)G213,14 (H-9GYEVGGQK16-NH2), and Aβ(9–16)G310,13,14 (H-9GGEVGGQK16-NH2), were manually synthesised in order to study Al binding to more specific amino acid residues. Both the peptides and the corresponding complexes with aluminium were comparatively investigated by mass spectrometry (MS), circular dichroism spectroscopy (CD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Al–peptide molecular ions and Al-fragment ions were unambiguously identified in the MS and MS/MS spectra. AFM images showed dramatic changes in the film morphology of peptides upon Al binding. Our findings from the investigation of N-terminal 1-16 and even 9-16 normal and modified sequences of Aβ peptides suggest that they have the capability to be involved in aluminium ion binding associated with AD.


Introduction
The prevalence of Alzheimer's disease (AD) continues to increase worldwide, becoming a great healthcare challenge of the twenty-first century [1]. AD is a progressive neurodegenerative disorder that gradually deprives the patient of cognitive function, being characterised by the presence of senile plaques and neurofibrillary tangles in the hippocampus and neocortex of the brain [2]. Intracranial amyloid aggregates, which are dependent on the proteolysis of the Aβ precursor protein (APP), are usually found in AD brains [3,4]. Such aggregates are made up of amyloid-β (Aβ) peptides, cholesterol, and metal ions and generate so-called neural plaques. The proteolytic Aβ peptides are normally degraded and removed, but they may accumulate in AD brains, mainly in presynaptic terminals of neurons to form insoluble plaques [5]. Peptide aggregates lead to the death of affected hippocampal neurons [6]. Aβ plaques contribute to neuronal loss and ultimate failure of cognitive function [7].

Peptide Synthesis
First, two modified peptides, analogues of the Aβ(1-16) peptide fragment, were synthesised by SPPS according to the Fmoc/t-Bu strategy on an automated peptide synthesiser. In the native Aβ(1- 16) peptide, which was also synthesised under the same conditions, the three histidine residues were replaced with alanine (Aβ(1-16)A3 6,13,14 :H-1 DAEFRADSGYEVAAQK 16 -NH2) and serine (Aβ(1- 16) S3 6,13,14 :H-1 DAEFRSDSGYEVSSQK 16 -NH2), in order to produce two peptides lacking the imidazole ring but with similar flexibility to the native Aβ(1- 16) peptide. The serine-rich peptide also contains more hydroxyl groups than the native peptide and could bind better aluminium ions. In addition, hydroxyl groups can induce the formation of hydrogen bonds that could lead to the aggregation and fibrillation of free serine peptides.
Second, the Aβ(9-16) peptide fragment and its three analogues, namely Aβ(9-16)G1 10 (H-9 GGEVHHQK 16 -NH2), Aβ(9-16)G2 13,14 (H-9 GYEVGGQK 16 -NH2), and Aβ(9-16)G3 10,13,14 (H-9 GGEVGGQK 16 -NH2), were manually synthesised on a Rink amide resin as solid support using the same fluorenylmetoxycarbonyl chemistry. The resulting crude peptides were purified by RP-HPLC chromatography on a C18 column. Here, Figure 1 refers only to the characterisation with an electrospray ionisation (ESI) ion trap mass spectrometer of the Aβ(1- 16) peptide, but all the other peptides used in our experiments were measured by the same procedure (not shown).   3+ were found at m/z 678.0 and m/z 690. 3. While a simple mass spectrometer determines the mass of a molecular ion based on the mass/electric charge ratio, tandem mass spectrometry (MS/MS) is used to select a particular molecular ion to determine the mass of the resulting fragment ions and a certain fragmentation pattern. If a molecular ion of a metal complex is fragmented, the fragment containing the metal ion can be stabilised and coordination sites can be determined by collision-induced dissociation (CID). The structure of Aβ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) was also investigated by tandem mass spectrometry (MS/MS), based on a collision-induced dissociation process [32]. The most intense peak in the mass spectrum, belonging to the [M + 3H] 3+ ion, was selected and subjected to MS/MS fragmentation (Figure 1b). The two intense peaks from m/z 542.1 and m/z 613.8 were assigned to the y-type fragments (y 9 2+ and y 15 3+ ). This type of fragmentation indicated the reduced stability of the peptide bond between the aspartic acid and alanine as well as serine residues. The presence of such peaks in the MS/MS spectra of peptides and their modification in the spectra of aluminium complexes of the investigated peptides may suggest not only the binding site of the metal ion, but also its influence upon the fragmentation mechanism in the CID process.
The unusual peaks were specific to the amidated C-terminal, the -CONH 2 group being prone to dehydration by the loss of a water molecule and the formation of a -CN functional group. The investigation of the MS/MS spectrum of a shorter peptide such as the Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptide fragment revealed a wealth of information about its structure. Typical ion fragments that may result from collision-induced dissociation are exemplified in Figure 2. The MS/MS spectrum in Figure 3 shows that the fragmentation occurred mainly in histidine residues; the highest peak was attributed to the y 3 + fragment resulting from the division of the two histidine residues. In addition, the y 4 + fragment visible at m/z 548.5 showed a rupture of the peptide bond between histidine and valine. However, while the presence of a b 5 + fragment ion confirms that the peptide bond between the two histidine residues can be broken, the relatively intense peaks of the b 6 + and b 7 + fragment ions may explain the formation of HQ and HH fragments. The m/z values calculated with GPMAW software confirmed both the structure and the stability of the peptide molecule.
to the y3 + fragment resulting from the division of the two histidine residues. In addition, the y4 + fragment visible at m/z 548.5 showed a rupture of the peptide bond between histidine and valine. However, while the presence of a b5 + fragment ion confirms that the peptide bond between the two histidine residues can be broken, the relatively intense peaks of the b6 + and b7 + fragment ions may explain the formation of HQ and HH fragments. The m/z values calculated with GPMAW software confirmed both the structure and the stability of the peptide molecule.  (9)(10)(11)(12)(13)(14)(15)(16) peptide, of which some may appear in the MS/MS spectra; (b) the structure of band y-type fragment ions of the GYEVHHQK-NH 2 peptide.
Following MS experiments with the Al-peptide complexes, we selected the parent ion [M + Al − 2H] + and subjected it to CID fragmentation. The assignment of the signals observed in the MS/MS spectra of [M + Al − 2H] + ions was performed by comparing the theoretical molecular masses of the ions resulting from the CID fragmentation with the experimentally recorded values. The (b) MALDI-ToF MS spectra of Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptides in the presence of aluminium ions (AlCl 3 ).
Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptides, containing two histidine residues in the molecule, generated numerous peaks, most of them bound to Al ions, such as [Al + x 6 Figure 7). Only a few peaks were assigned to peptide fragment ions without aluminium. The Aβ (9-16) G 1 10 peptide, obtained by replacing the tyrosine residue with glycine, also had many peaks in the MS/MS spectrum, which were attributed to Al-containing peptide fragment ions such as [Al + a 6 suggesting that aluminium protected the peptide bond between the two histidine residues.
In addition, while the fragmentation of the free peptides occurred mainly at the peptide bond between the histidine residues to form the y 3 + fragment, Al induced different fragmentation to generate the a-type fragments containing metal ions.
Aβ ( Aβ(9-16) peptides, containing two histidine residues in the molecule, generated numerous peaks, most of them bound to Al ions, such as [Al + x6 (Figure 7). Only a few peaks were assigned to peptide fragment ions without

Fourier Transform Infrared Spectroscopy (FT-IR)
The conformational changes undergone by the peptides in the presence of aluminium ions were also investigated by FT-IR (  As for the glycine-enriched peptides, only the y 4 + and y 5 + fragment ions were found to possess Al bound to their backbone, which suggests a weaker interaction of these peptides with Al ions. Besides, the presence of [b 4 -NH 2 ] + and [b 5 -NH 2 ] + structures was noticed in the MS/MS spectrum of the Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptide and [c 5 -NH 2 ] + in that of the Aβ (9-16) G 2 13,14 peptide, which suggests a possible cyclisation followed by removing the NH 2 radical induced by Al. The main peak in the MS/MS spectrum of the Aβ (9-16) G 3 10,13,14 peptide was assigned to z 7 + , while this fragment does not appear in the spectrum of free peptides. This may be evidence that the binding of Al to the investigated peptides changed their stability and induced different fragmentation patterns. Although aluminium ions may be not capable of binding to the histidine residues of Aβ peptides, they can bind easily to HHQK peptide fragments and less well to GYEV or GGEV residues. However, glycine residues in the modified Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptides induce a high degree of flexibility, which may inhibit Al binding to peptides.

Fourier Transform Infrared Spectroscopy (FT-IR)
The conformational changes undergone by the peptides in the presence of aluminium ions were also investigated by FT-IR ( The FT-IR spectra of the four peptides showed characteristic bands for the individual peptides, but each of them exhibited a relatively high peak at 722 cm −1 . If the Aβ(9-16) and Aβ(9-16)G1 10 peptides had absorption bands at 627 cm −1 and 629 cm −1 , respectively, these peaks disappeared from the FT-IR spectra of glycine-rich peptides, probably due to the removal of the imidazole ring. Upon adding Al, the 400-725 cm −1 region changed dramatically. Two large and high-intensity bands appeared due to Al-O and Al-N bonds, one of them at 520-528 cm −1 and the other one at 577 cm −1 . Another large band appeared at 832-838 cm −1 , being generated from the two narrow peaks 799-800 cm −1 and 835-838 cm −1 . The presence of aluminium ions also determined a decrease in the intensity of the signal found in the range of 1100-1200 cm −1 , associated with the bending vibrations of C-C-N bonds, as well as the stretching vibrations of C-N bonds. In addition, the influence of Al 3+ ions was observed in the maximum values of absorption, where displacements at longer wavelengths by about 10 units were identified: Aβ(9-16) from 1134 cm −1 to 1146 cm −1 , Aβ(9-16)G1 10 from 1134 cm −1 to 1144 cm −1 , Aβ(9-16)G2 13,14 from 1135 cm −1 to 1148 cm −1 , and Aβ(9-16)G3 10,13,14 from 1133 cm −1 to 1148 cm −1 . A strong influence was also noticed in the region of 1400-1450 cm −1 , where the signal corresponding to the asymmetric stretching vibrations of the CH3 group, of the deformation in the plane of the CH2 group, and of the stretching of the CN bond underwent a partial division that led to the appearance of several peaks.
In the interval of 1500-1700 cm −1 , specific to the amide I and amide II bonds, the aluminium ions determined a partial overlap of the two absorption maxima. Specifically, the maximum values recorded in the spectra of the Aβ(9-16) peptides (1538 cm −1 ) in the presence of metal ions were attributed to disordered structures, the influence of β-conformation folding observed in the spectra of free peptides being much diminished. Similar changes were observed in the case of glycine-enriched peptides (Aβ(9-16)G1 10 , Aβ(9-16)G2 13,14 , and Aβ(9-16)G3 10,13,14 ), where the presence of aluminium favoured the adoption of rather disordered structures, but α-helix and β-sheet conformers may also have been present. Nevertheless, it was difficult to see the presence of all types of conformers among the small peptide molecules from the FT-IR spectra. The FT-IR spectra of the four peptides showed characteristic bands for the individual peptides, but each of them exhibited a relatively high peak at 722 cm −1 . If the Aβ (9)(10)(11)(12)(13)(14)(15)(16) and Aβ (9-16) G 1 10 peptides had absorption bands at 627 cm −1 and 629 cm −1 , respectively, these peaks disappeared from the FT-IR spectra of glycine-rich peptides, probably due to the removal of the imidazole ring. Upon adding Al, the 400-725 cm −1 region changed dramatically. Two large and high-intensity bands appeared due to Al-O and Al-N bonds, one of them at 520-528 cm −1 and the other one at 577 cm −1 . Another large band appeared at 832-838 cm −1 , being generated from the two narrow peaks 799-800 cm −1 and 835-838 cm −1 . The presence of aluminium ions also determined a decrease in the intensity of the signal found in the range of 1100-1200 cm −1 , associated with the bending vibrations of C-C-N bonds, as well as the stretching vibrations of C-N bonds. In addition, the influence of Al 3+ ions was observed in the maximum values of absorption, where displacements at longer wavelengths by about 10 units were identified: Aβ (9)(10)(11)(12)(13)(14)(15)(16) from 1134 cm −1 to 1146 cm −1 , Aβ (9-16) G 1 10 from 1134 cm −1 to 1144 cm −1 , Aβ (9-16) G 2 13,14 from 1135 cm −1 to 1148 cm −1 , and Aβ (9-16) G 3 10,13,14 from 1133 cm −1 to 1148 cm −1 .
A strong influence was also noticed in the region of 1400-1450 cm −1 , where the signal corresponding to the asymmetric stretching vibrations of the CH 3 group, of the deformation in the plane of the CH 2 group, and of the stretching of the CN bond underwent a partial division that led to the appearance of several peaks.
In the interval of 1500-1700 cm −1 , specific to the amide I and amide II bonds, the aluminium ions determined a partial overlap of the two absorption maxima. Specifically, the maximum values recorded in the spectra of the Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptides (1538 cm −1 ) in the presence of metal ions were attributed to disordered structures, the influence of β-conformation folding observed in the spectra of free peptides being much diminished. Similar changes were observed in the case of glycine-enriched peptides (Aβ (9-16) G 1 10 , Aβ (9-16) G 2 13,14 , and Aβ (9-16) G 3 10,13,14 ), where the presence of aluminium favoured the adoption of rather disordered structures, but α-helix and β-sheet conformers may also have been present. Nevertheless, it was difficult to see the presence of all types of conformers among the small peptide molecules from the FT-IR spectra. Structural changes were also identified in the amide I domain, where the signal displayed in the spectra of peptides incubated with Al 3+ ions showed a single maximum value, which suggested a β-folded structure (1631 cm −1 , 1634 cm −1 , and 1637 cm −1 ), unlike the spectra of free peptides, which suggested mainly α-helical conformations (Aβ (9-16) G 2 13,14 and Aβ (9-16) G 3 10,13,14 ). Figure 9 shows the spectra of second-order derivatives of amyloid peptides in the absence and presence of aluminium ions. The calculation of the derivative made it possible to obtain more accurate information in the amide I region on the conformations adopted by the peptides. Although the β-folded structure of the peptides Aβ (9)(10)(11)(12)(13)(14)(15)(16) and Aβ (9)(10)(11)(12)(13)(14)(15)(16) G 1 10 was also present when they were treated with AlCl 3 , by generating signals in the 1639-1630 cm −1 region, the most important result was the intense bands with maxima at 1612-1613 cm −1 , attributed to an aggregate-type structure (Figure 9b). However, the α-helical conformation was evidenced in the structure of the peptides Aβ (9-16) G 2 13,14 and Aβ (9)(10)(11)(12)(13)(14)(15)(16)  Structural changes were also identified in the amide I domain, where the signal displayed in the spectra of peptides incubated with Al 3+ ions showed a single maximum value, which suggested a βfolded structure (1631 cm −1 , 1634 cm −1 , and 1637 cm −1 ), unlike the spectra of free peptides, which suggested mainly α-helical conformations (Aβ(9-16)G2 13,14 and Aβ(9-16)G3 10,13,14 ). Figure 9 shows the spectra of second-order derivatives of amyloid peptides in the absence and presence of aluminium ions. The calculation of the derivative made it possible to obtain more accurate information in the amide I region on the conformations adopted by the peptides. Although the βfolded structure of the peptides Aβ(9-16) and Aβ(9-16)G1 10 was also present when they were treated with AlCl3, by generating signals in the 1639-1630 cm −1 region, the most important result was the intense bands with maxima at 1612-1613 cm −1 , attributed to an aggregate-type structure (Figure 9b). However, the α-helical conformation was evidenced in the structure of the peptides Aβ(9-16)G2 13,14 and Aβ(9-16)G3 10,13,14

Atomic Force Microscopy
AFM images showed that peptide assemblies have different two-dimensional layer-shaped structures ( Figure 10). A solution of free Aβ(1-16) peptides produced a smooth film and no fibrils or aggregates were evident on the glass plate of the microscope. We assumed that its low tendency to aggregation is related to the hydrophilic N-terminus sequence of amyloid-β peptides. On the contrary, the alanine-rich Aβ(1-16) peptide formed fibrils, most probably due to its hydrophobic characteristic, while serine-rich peptides had a pronounced tendency to fibrillation due to hydrogen bonding of serine residues. Thus, by replacing the histidine residues with alanine in the native Aβ(1- AFM images showed that peptide assemblies have different two-dimensional layer-shaped structures ( Figure 10). A solution of free Aβ (1-16) peptides produced a smooth film and no fibrils or aggregates were evident on the glass plate of the microscope. We assumed that its low tendency to aggregation is related to the hydrophilic N-terminus sequence of amyloid-β peptides. On the contrary, the alanine-rich Aβ (1-16) peptide formed fibrils, most probably due to its hydrophobic characteristic, while serine-rich peptides had a pronounced tendency to fibrillation due to hydrogen bonding of serine residues. Thus, by replacing the histidine residues with alanine in the native Aβ (1-16) sequence, a texture composed of differently sized fibrils appeared. Some fibrils were about 80-100 nm thick and more than 0.5 µm long, whereas the other ones were smaller but also in the nanometre range. We supposed that alanine residues highly increased the peptide hydrophobicity, as well as its α-helical proportion, thus making possible the association of molecules through non-covalent interactions. Serine-rich Aβ (1-16) peptides generated even larger fibrils and aggregates. This phenomenon could be possible due to the intermolecular hydrogen bonds. AFM images showed that peptide assemblies have different two-dimensional layer-shaped structures ( Figure 10). A solution of free Aβ(1-16) peptides produced a smooth film and no fibrils or aggregates were evident on the glass plate of the microscope. We assumed that its low tendency to aggregation is related to the hydrophilic N-terminus sequence of amyloid-β peptides. On the contrary, the alanine-rich Aβ(1-16) peptide formed fibrils, most probably due to its hydrophobic characteristic, while serine-rich peptides had a pronounced tendency to fibrillation due to hydrogen bonding of serine residues. Thus, by replacing the histidine residues with alanine in the native Aβ(1- 16) sequence, a texture composed of differently sized fibrils appeared. Some fibrils were about 80-100 nm thick and more than 0.5 μm long, whereas the other ones were smaller but also in the nanometre range. We supposed that alanine residues highly increased the peptide hydrophobicity, as well as its α-helical proportion, thus making possible the association of molecules through non-covalent interactions. Serine-rich Aβ(1-16) peptides generated even larger fibrils and aggregates. This phenomenon could be possible due to the intermolecular hydrogen bonds.  In brief, AFM images, as well as CD and FT-IR spectra, showed that the Aβ (1-16) peptide conformation and fibrillation depend on the peptide sequence and the presence of aluminium in solution. The film surface of the native peptide Aβ (1-16) changed only slightly with the addition of aluminum at pH 6.6 ( Figure 9b). Aluminium ions induced the aggregation and fibrillation of modified peptides as demonstrated by AFM and SEM experiments. Thus, Aβ (1-16) A 3 6,13,14 generated fibrils even in the absence of Al, being a hydrophobic peptide. In the presence of Al sulphate, the fibrils became longer and thicker. The excess of aluminium salt can be observed in the AFM images for the 1:2 peptide:Al ratio. On the contrary, serine-rich peptides can form fibrils due to the hydrogen bonds.

Scanning Electron Microscopy
The interaction of the two modified Aβ (1- 16) peptides with aluminium was confirmed by SEM ( Figure 11) that showed a rough surface, with fibrils differently and densely distributed. As the concentration of metal ions increased, the large fibrils formed (Figure 10e), became longer, and some of them became thinner (Figure 10f). We assumed that Al increased the hydrophobicity of Aβ (1-16) A 3 6,13,14 , which led to the formation of thicker and longer fibrils, followed by their division as aluminium sulphate concentration increased. In addition, these fibrils are longer and intertwined. The serine-rich peptides show short, thin fibres, which thickened and lengthened in the presence of aluminium ions (Figure 11c,d).

Nuclear Magnetic Resonance Spectroscopy
In the NMR study of the Aβ (1-16) and Aβ (9)(10)(11)(12)(13)(14)(15)(16) fragments as well as their analogues, presented here, the interactions of these peptides with aluminium were investigated. Although we found significant changes of 1 H-NMR spectra in the presence of aluminium sulphate, we investigate here only the spectra of some very small peptides as models. We were interested in showing that the amino acid residues may be capable of binding to or interacting with aluminium ions in solutions. However, we present here, in Figure 14, some spectra of Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptide fragments in deuterium oxide solution, alone, or in the presence of aluminium ions.
Our experiment showed that glycyl-tyrosine bound aluminium ions in the aluminium sulphate aqueous solutions at the peptide bond region and not at the phenolic OH group (Figure 12). Although all the protons were affected by Al, the most influenced were the protons of the tyrosine CH 2 group (the peaks shifted from 3.085-3.123 ppm to 3.125-3.212 ppm, also becoming enlarged) and the protons of the glycine CH 2 group (from 3.630-3.769 ppm to 3.661-3.800 ppm). Of course, the proton near the COOH group was also much affected.
occur in the case of the formation of Aβ(1-16)A3 films in the presence of aluminium ions ( Figure  10e,f). As the concentration of metal ions increased, the large fibrils formed (Figure 10e), became longer, and some of them became thinner (Figure 10f). We assumed that Al increased the hydrophobicity of Aβ(1-16)A3 6,13,14 , which led to the formation of thicker and longer fibrils, followed by their division as aluminium sulphate concentration increased. In addition, these fibrils are longer and intertwined. The serine-rich peptides show short, thin fibres, which thickened and lengthened in the presence of aluminium ions (Figure 11c,d).

Nuclear Magnetic Resonance Spectroscopy
In the NMR study of the Aβ(1-16) and Aβ(9-16) fragments as well as their analogues, presented here, the interactions of these peptides with aluminium were investigated. Although we found significant changes of 1 H-NMR spectra in the presence of aluminium sulphate, we investigate here only the spectra of some very small peptides as models. We were interested in showing that the amino acid residues may be capable of binding to or interacting with aluminium ions in solutions. However, we The 1 H-NMR spectrum of seryl-glycine contains the serine CH 2 protons in the 3.754-3.897 ppm region ( Figure 13). Under the influence of Al, we found the peaks shifted to 3.799-3.927 ppm, but the distance between the highest signals was smaller. These findings demonstrate that aluminium ions are bound to serine OH groups. In addition, the two protons of the glycine CH 2 group were found to be shifted toward higher ppm values and wider. Besides, the CH groups of serine residues bound to amino groups were also modified.
Our experiment showed that glycyl-tyrosine bound aluminium ions in the aluminium sulphate aqueous solutions at the peptide bond region and not at the phenolic OH group (Figure 12). Although all the protons were affected by Al, the most influenced were the protons of the tyrosine CH2 group (the peaks shifted from 3.085-3.123 ppm to 3.125-3.212 ppm, also becoming enlarged) and the protons of the glycine CH2 group (from 3.630-3.769 ppm to 3.661-3.800 ppm). Of course, the proton near the COOH group was also much affected. The 1 H-NMR spectrum of seryl-glycine contains the serine CH2 protons in the 3.754-3.897 ppm region ( Figure 13). Under the influence of Al, we found the peaks shifted to 3.799-3.927 ppm, but the distance between the highest signals was smaller. These findings demonstrate that aluminium ions are bound to serine OH groups. In addition, the two protons of the glycine CH2 group were found to be shifted toward higher ppm values and wider. Besides, the CH groups of serine residues bound to amino groups were also modified. These data confirm that the peptide backbone is also involved in aluminium binding and the hydroxyl group of serine also binds aluminium ions. Figure 14 shows that the NMR spectra of the Aβ(9-16) peptide is slightly changed in the presence of aluminium ions. However, we also studied by NMR spectroscopy Al binding to Aβ(9-16), Aβ(1-16), or only its interaction with these peptides, as well as their modified variants, and continued with Aβ(1- 40) and its variants in which the histidine residues were replaced with serine and alanine ones (not shown). Some other experiments are in progress, in which larger amounts of peptides are used, and, therefore, a new manuscript is expected to disseminate the results. These data confirm that the peptide backbone is also involved in aluminium binding and the hydroxyl group of serine also binds aluminium ions. Figure 14 shows that the NMR spectra of the Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptide is slightly changed in the presence of aluminium ions. However, we also studied by NMR spectroscopy Al binding to Aβ (9)(10)(11)(12)(13)(14)(15)(16) , Aβ (1- 16) , or only its interaction with these peptides, as well as their modified variants, and continued with Aβ (1-40) and its variants in which the histidine residues were replaced with serine and alanine ones (not shown). Some other experiments are in progress, in which larger amounts of peptides are used, and, therefore, a new manuscript is expected to disseminate the results.

Discussion
Interaction of metal ions with Aβ is mediated mostly by the N-terminal Aβ(1−16) domain and appears to play an important role in AD progression [36]. Although Al ions also were reported to bind to some residues found in the Aβ(1-16) sequence, no measurable amounts of Aβ(1-28)-Al 3+ adducts were noticed by other authors within their experiments by 1 H NMR and electrospray ionisation mass spectrometry (ESI-MS) at physiological pH [37]. These were the incentive conditions for the present investigation of Al interactions with Aβ in which we synthesised several variants of the Aβ(1-16) and Aβ(9-16) peptides, and which contain histidine residues replaced by alanine, serine, or glycine. Since the interaction of amyloid-β peptides with Al ions might be involved in AD pathogenesis, we investigated aluminium binding to the newly synthesised peptides by MS, FT-IR, CD, AFM, and NMR spectroscopy.
Coordination of Al ions to the N-terminus of Aβ at the acidic residues Glu 3 , Asp 7 , and Glu 11 induces significant helical content, which means a major impact on the structure and dynamics of the peptide and reducing the impact of salt bridges, as well as the flexibility of binding residues and increasing that of terminal residues [23]. High helical content and disruption of salt bridges lead to a characteristic tertiary structure, as shown by heat maps of contact between residues, as well as representative clusters of trajectories [23]. The Al-induced flexibility allows for significant quantities of helical secondary structure to develop. Indeed, such metal ions affect residues 11-20 and 26-36 [23]. Salt bridges are strongly affected by the presence of the Al ion in the 11-16 region, as we demonstrated here. This, along with hydrogen bond patterns of serine-rich peptides that reflect the rather high helical content, lead to characteristic patterns in the tertiary structure in which stable contacts between salt-bridged pairs, as well as residues bound to Al ions, are apparent. Figure 14. 1 H-NMR spectra of Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptide in the presence of aluminium sulphate: (a) COSY 1 H NMR spectrum of Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptide; (b) COSY 1 H NMR spectrum of Aβ (9)(10)(11)(12)(13)(14)(15)(16) in the presence of Al 3+ ; (c) 1 H-NMR spectra.

Discussion
Interaction of metal ions with Aβ is mediated mostly by the N-terminal Aβ (1−16) domain and appears to play an important role in AD progression [36]. Although Al ions also were reported to bind to some residues found in the Aβ (1-16) sequence, no measurable amounts of Aβ (1-28) -Al 3+ adducts were noticed by other authors within their experiments by 1 H NMR and electrospray ionisation mass spectrometry (ESI-MS) at physiological pH [37]. These were the incentive conditions for the present investigation of Al interactions with Aβ in which we synthesised several variants of the Aβ (1-16) and Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptides, and which contain histidine residues replaced by alanine, serine, or glycine. Since the interaction of amyloid-β peptides with Al ions might be involved in AD pathogenesis, we investigated aluminium binding to the newly synthesised peptides by MS, FT-IR, CD, AFM, and NMR spectroscopy.
Coordination of Al ions to the N-terminus of Aβ at the acidic residues Glu 3 , Asp 7 , and Glu 11 induces significant helical content, which means a major impact on the structure and dynamics of the peptide and reducing the impact of salt bridges, as well as the flexibility of binding residues and increasing that of terminal residues [23]. High helical content and disruption of salt bridges lead to a characteristic tertiary structure, as shown by heat maps of contact between residues, as well as representative clusters of trajectories [23]. The Al-induced flexibility allows for significant quantities of helical secondary structure to develop. Indeed, such metal ions affect residues 11-20 and 26-36 [23]. Salt bridges are strongly affected by the presence of the Al ion in the 11-16 region, as we demonstrated here. This, along with hydrogen bond patterns of serine-rich peptides that reflect the rather high helical content, lead to characteristic patterns in the tertiary structure in which stable contacts between salt-bridged pairs, as well as residues bound to Al ions, are apparent.
Therefore, FT-IR and CD are convenient methods to predict metal binding and conformational changes.
Although AD is not curable, some treatments are available to slow down the disease progression and reduce the cognitive impairment and behavioural problems, but do not stop the progression of neurodegeneration [38]. Our research may show new details about Aβ aggregation and fibrillation associated with AD and highlight the role of aluminium ions in creating a hydrophobic environment that increases the tendency of Aβ peptides to form plaques in AD brains.
The presented results are in good agreement with recent data demonstrating that the topology of Aβ (1-16) chelated with the Al 3+ ion remains preserved, as compared to the free peptide [39], while another study performed with the Aβ (1-28) fragment revealed no measurable amounts of Al-peptide adducts upon peptide-metal mixing, regardless of experimental conditions [37]. We also considered that histidine residues are not involved in Al binding, but they may influence aluminium binding due to the rigidity of the peptide backbone.
The process of minimising peptide energy in silico was performed involving steric energy ( Figure 15). For this study, the modified peptide Aβ (1-16) S 3 6,13,14 was taken into consideration to understand the influence of the Al 3+ ion on its secondary structure. Because serine residues introduced into the native Aβ (1-16) due to the hydroxyl group can form specific bonds with aluminium ions, we analysed the influence of these functional groups in complementarity with other amino and hydroxyl functions in the Aβ (1-16) secondary structure. We assumed that the two oxygen atoms of the hydroxyl groups of the two serine residues at positions 13 and 14 (atoms no. 24 and 30) bind to the Al 3+ ion, while a third bond is formed between Al and the atom of oxygen or nitrogen from the amino and hydroxyl functional groups (numbered according to Figure 15a). Thus, we found 11 possible variants of such an interaction with Al. Each version was applied to the process of minimising energy in order to obtain the most stable conformation from the energy point of view.
Molecules 2020, 25, x FOR PEER REVIEW 18 of 26 Therefore, FT-IR and CD are convenient methods to predict metal binding and conformational changes.
Although AD is not curable, some treatments are available to slow down the disease progression and reduce the cognitive impairment and behavioural problems, but do not stop the progression of neurodegeneration [38]. Our research may show new details about Aβ aggregation and fibrillation associated with AD and highlight the role of aluminium ions in creating a hydrophobic environment that increases the tendency of Aβ peptides to form plaques in AD brains.
The presented results are in good agreement with recent data demonstrating that the topology of Aβ(1-16) chelated with the Al 3+ ion remains preserved, as compared to the free peptide [39], while another study performed with the Aβ(1-28) fragment revealed no measurable amounts of Al-peptide adducts upon peptide-metal mixing, regardless of experimental conditions [37]. We also considered that histidine residues are not involved in Al binding, but they may influence aluminium binding due to the rigidity of the peptide backbone.
The process of minimising peptide energy in silico was performed involving steric energy ( Figure 15). For this study, the modified peptide Aβ(1-16)S3 6,13,14 was taken into consideration to understand the influence of the Al 3+ ion on its secondary structure. Because serine residues introduced into the native Aβ(1-16) due to the hydroxyl group can form specific bonds with aluminium ions, we analysed the influence of these functional groups in complementarity with other amino and hydroxyl functions in the Aβ(1-16) secondary structure. We assumed that the two oxygen atoms of the hydroxyl groups of the two serine residues at positions 13 and 14 (atoms no. 24 and 30) bind to the Al 3+ ion, while a third bond is formed between Al and the atom of oxygen or nitrogen from the amino and hydroxyl functional groups (numbered according to Figure 15a). Thus, we found 11 possible variants of such an interaction with Al. Each version was applied to the process of minimising energy in order to obtain the most stable conformation from the energy point of view. Molecular modelling indicated that the site of aluminium binding migrates toward the Cterminus of the peptide, and the peptide backbone tends to adopt a rather β-sheet conformation, thus reducing the proportion of α-helix conformers that induced the specific elasticity of the peptide. Therefore, these findings suggest that with the interaction of aluminium ions with nitrogen and oxygen atoms, the rigidity of the structure increases, thus giving the peptide an attribute of inaccessibility to solvents, according to Figure 16. This may also suggest an increase in the hydrophobicity of the serine-rich peptide, which induces an increased tendency to aggregate. Nevertheless, we should keep in mind that a large proportion of Aβ molecules are free and that a large amount of Al is unbound, acting in the form of inorganic compounds. These theoretical results confirm experimental CD, AFM, and FT-IR data. Molecular modelling indicated that the site of aluminium binding migrates toward the C-terminus of the peptide, and the peptide backbone tends to adopt a rather β-sheet conformation, thus reducing the proportion of α-helix conformers that induced the specific elasticity of the peptide. Therefore, these findings suggest that with the interaction of aluminium ions with nitrogen and oxygen atoms, the rigidity of the structure increases, thus giving the peptide an attribute of inaccessibility to solvents, according to Figure 16. This may also suggest an increase in the hydrophobicity of the serine-rich peptide, which induces an increased tendency to aggregate. Nevertheless, we should keep in mind that a large proportion of Aβ molecules are free and that a large amount of Al is unbound, acting in the form of inorganic compounds. These theoretical results confirm experimental CD, AFM, and FT-IR data.

Materials and Methods
All chemicals were of analytical or higher grade and purchased from well-recognised commercial sources. The solvents for peptide synthesis were commercial analytical grade and were redistilled before use. All solutions and buffers were prepared using MilliQ-grade water (18.2 MΩ·cm) from a Millipore water purification system (Milford, MA, USA). As solid support for the peptide synthesis, a Rink amide resin procured from Sigma-Aldrich Chemie GmbH was used (Taufkirchen, Germany). The following side chain-protected amino acids were used . Piperidine (PYP) and bromophenol blue were obtained from Merck (Germany), while bradykinin, substance P, renin, adrenocorticotropin (ACTH), and oxidized insulin B-chain were from Sigma, city, Germany.
In addition to the peptides belonging to the Aβ(1-16) and Aβ(9-16) group, which were synthesised in our laboratory, other peptides and an amino acid were associated with the experiments of aluminium binding. The dipeptide glycyl-tyrosine (Gly-Tyr) was supplied by Fluka Chem. Co. (Steinheim, Germany) and chosen as a model for the 9 GY 10 peptide fragment in the binding of aluminium ions. In addition, another dipeptide, seryl-glycine (Sigma), was used to study the behaviour of the system 8 SG 9 . Because the C-terminus of these two peptides is not amidated or protected, we also introduced in our experiments tyrosinamide (Tyr-NH2) obtained from Sigma-Aldrich.

Materials and Methods
All chemicals were of analytical or higher grade and purchased from well-recognised commercial sources. The solvents for peptide synthesis were commercial analytical grade and were redistilled before use. All solutions and buffers were prepared using MilliQ-grade water (18.2 MΩ·cm) from a Millipore water purification system (Milford, MA, USA). As solid support for the peptide synthesis, a Rink amide resin procured from Sigma-Aldrich Chemie GmbH was used (Taufkirchen, Germany). . Piperidine (PYP) and bromophenol blue were obtained from Merck (Germany), while bradykinin, substance P, renin, adrenocorticotropin (ACTH), and oxidized insulin B-chain were from Sigma, city, Germany.
In addition to the peptides belonging to the Aβ (1-16) and Aβ (9)(10)(11)(12)(13)(14)(15)(16) group, which were synthesised in our laboratory, other peptides and an amino acid were associated with the experiments of aluminium binding. The dipeptide glycyl-tyrosine (Gly-Tyr) was supplied by Fluka Chem. Co. (Steinheim, Germany) and chosen as a model for the 9 GY 10 peptide fragment in the binding of aluminium ions. In addition, another dipeptide, seryl-glycine (Sigma), was used to study the behaviour of the system 8 SG 9 . Because the C-terminus of these two peptides is not amidated or protected, we also introduced in our experiments tyrosinamide (Tyr-NH 2 ) obtained from Sigma-Aldrich.
Analytical RP-HPLC was also performed on a SpectraSystem using an analytical Vydac C8 column (250 mm × 4.6 mm) with 5 µm silica (300 Å pore size) as a stationary phase (Grace Columns). The method was run at 1.0 mL min −1 using a mobile phase similar to the semi-preparative method.

MALDI-ToF Mass Spectrometry
Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) analysis was carried out with a Bruker Ultraflex MALDI ToF/ToF mass spectrometer operated in positive reflectron mode and equipped with a pulsed nitrogen UV laser (Bruker Daltonics, Hamburg, Germany). For this analysis, the samples were co-crystallised with an excess of organic matrix capable of absorbing at 337 nm and volatilising under the action of laser radiation. As a matrix, a saturated solution of α-cyano-4-hydroxy-cinnamic acid (CHCA) dissolved in a solution containing 2:1 ACN:0.1% TFA in MilliQ was used for peptide mapping. Using the dried drop method, which implies adding first 1 µL of sample and over it 1 µL of freshly prepared matrix solution, the mixture was deposited on a conductive metallic plate called a target and allowed to dry. After co-crystallisation, the metal plate was introduced into the mass spectrometer and bombarded with short laser pulses. The desorbed and ionised molecules were accelerated by an electrostatic field and discharged through a high-fly metal flight tube. Depending on their mass, ionised molecules reached the detector at different times.
The spectra were recorded in positive reflectron mode using the following parameters: 20 kV acceleration voltage, 40% grid voltage, 140 ns delay, low-mass gate of 500 Da, and an acquisition mass range of 600-3500 Da. The final mass spectrum represented an accumulation of 300 shots per acquisition. The obtained spectra were processed using Bruker's Flex Analysis 3.4 software.
of each sample (approximately 50 µL of sample solution) were allowed to dry overnight on small glass slides in dust-free medium (covered with a Petri dish), at room temperature.

Scanning Electron Microscopy
A scanning electron microscope (SEM; Ultra plus Scanning Electron Microscope from Carl Zeiss NTS) (Manufacturer, City, State abbr. if USA, Country), operating at 4 kV with secondary electrons, in high-vacuum mode, was used to observe the morphological properties of the Al-peptide complexes. The SEM studies were performed on samples dried on small glass slides, fixed on copper supports using carbon tape, and covered with a thin layer of platinum to avoid electrostatic charging. We used SEM to characterise the fibril morphology of aggregates formed by Aβ(1-16)A 3 6,13,14 and Aβ(1-16)S 3 6,13,14 upon incubation with aluminium sulphate (peptide concentration 256 µM; 1:1 molar ratio) for 24 h at 25 • C.

Fourier Transform Infrared Spectroscopy
The infrared spectra were measured using a Shimadzu 8400S FT-IR spectrophotometer (Shimadzu, Japan). The FT-IR spectra were recorded from 4000 to 400 cm −1 in spectroscopic-grade CsI with a detector at 2 cm −1 resolution and 20 scans per sample working in transmission mode. The collected FT-IR spectra were compared with the standard spectra of the functional groups.

NMR Spectroscopy
Proton NMR data were recorded by Dr. Catalina Ionica Ciobanu on a Bruker Avance III, 500 MHz frequency spectrometer, equipped with a 5 mm PABBO detection probe and operating at 500. 19 MHz for 1 H nucleus. Chemical shifts are reported in δ units (ppm), relative to the solvent residual peak (D 2 O, 1 H:4.79 ppm). Coupling constants are reported in Hertz (Hz). The number of scans for 1 H-NMR experiments was 256 and four scans for two-dimensional 1 H-1 H NMR spectra. The peptide sample was prepared in D 2 O as a solvent. The experiment was recorded without the introduction of a presaturation pulse for solvent signal suppression.

Data Analysis
The mass spectrometric data analyses were performed using the software Bruker Daltonics DataAnalysis 3.3. The molecular weight determination of peptides, as well as the prediction of the corresponding fragments in the MS/MS process of peptides and Al-fragment adducts, were conducted using GPMAW 6.11 software (General Protein/Mass Analysis for Windows, Lighthouse Data, Odense M, Denmark) [42]. The monoisotopic peak list was compared against data calculated by GPMAW. AFM image analysis was done with scanning probe microscopy software, WSxM 4.0 Develop 10.0 (NT-MTD Co. Zelenograd, Moscow, Russia). The obtained data following IR spectroscopy were processed using Origin software. Computer simulation was performed with the help of the Chem3D Ultra 10.0 program.

Conclusions
Our findings suggest that the N-terminal 1-16 sequence of Aβ peptides has the is involved in aluminium ion binding associated with AD. The key observation in our study is that aluminium ions interact with the N-terminus Aβ (1-16) sequence of amyloid-β peptides. In addition, the Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptide fragment also interacts with Al, as the FT-IR experiments suggest. Consequently, several variants of Aβ (1-16) and Aβ (9)(10)(11)(12)(13)(14)(15)(16) peptide fragments were synthesised and used to study their interaction with aluminium ions. These ions were suspected to induce the formation of Aβ fibrils and aggregates. Through the formation of metal ion adducts during the MALDI-ToF MS measurements, we used MS/MS spectra to investigate aluminium binding to amyloid-β peptide fragments and analogues, and to identify the most probable binding sites. AFM images, as well as CD and FT-IR spectra, showed that the Aβ (1-16) peptide conformation and fibrillation depend on the sequence of peptides and the presence of aluminium in solution. Free Aβ (1- 16) peptides, which are the hydrophilic N-terminus sequences of amyloid-β peptides, generated a smooth film surface because of their low tendency to aggregation. Instead, alanine-rich Aβ (1-16) formed fibrils, most probably due to its hydrophobic characteristic, while serine-rich peptides had a pronounced tendency to fibrillation, probably due to the hydrogen bonding of serine residues. Aluminium ions induce peptide aggregation and fibrillation, as demonstrated by AFM and SEM experiments. However, Al may have a higher affinity to amyloid peptides at a lower pH, and its binding could be pH dependent. Investigation of such short amyloid-β peptide fragments and analogues may provide clues for plaque formation under aggregation conditions and may facilitate the design of potential drugs for these targets. However, further research is needed to better understand the multiple interactions of aluminium ions with Aβ peptides for Alzheimer's disease.