*2.3. L17A*/*F19A Mutation Inhibits the Aggregation of A*β*40(E22Q)*

We characterized the effect of L17A/F19A mutation on the structural propensity of Aβ40(E22Q). However, the effect of L17A/F19A mutation on the aggregative property of Aβ40(E22Q) remained unclear. To investigate this issue, we applied thioflavin-T (Th-T) fluorescence assay and transmission electron microscopy (TEM) to monitor the aggregation processes of Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q) in aqueous solution. The results of Th-T assay and TEM are shown in Figures 7 and 8, respectively. It can be seen from Figure 7 that the shapes of the aggregation profiles of Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q) in aqueous solution are sigmoidal, suggesting that both peptides aggregated in a nucleation-dependent polymerization manner. Furthermore, the two aggregation profiles shown in Figure 7 displayed two distinct lag phases (nucleation phases) whose durations are 12 and 27 h for Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q), respectively. This result revealed that Aβ40(E22Q) aggregated more rapidly than Aβ40(L17A/F19A/E22Q). Figure 8 shows the TEM images of Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q) in aqueous solution acquired at different time points. Fibrils were observed at Day 1 and Day3 for Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q), respectively. This observation indicated that the rate of fibril formation is more rapid for Aβ40(E22Q) than for Aβ40(L17A/F19A/E22Q). Taken together, these findings suggested that the L17A/F19A mutation would reduce the aggregation rate of Aβ40(E22Q). From a kinetic point of view, the free energy of activation for conformational change from α-helix to β-strand would be higher for a peptide which adopts a higher α-helical propensity. Since the conformational change from the α-helix to the β-strand of Aβ is one of the key factors in governing its aggregative propensity, it is reasonable to infer that L17A/F19A mutation inhibits the aggregation of Aβ40(E22Q). This might be through increasing the α-helical propensity of its α/β-discordant region, which in turn reduces its rate of conformational change from the α-helix to the β-strand.

**Figure 7.** Aggregation kinetics of Aβ40(E22Q) (red) and Aβ40(L17A/F19A/E22Q) (black).

**Figure 8.** TEM images of Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q). The scale bar is 200 nm.

### **3. Discussion**

Recently, Hatami et al. reported the effects of FAD-related mutations within the Aβ sequence on the fibrils morphology and aggregation kinetics of Aβ using TEM and Th-T assay [11]. They found that most FAD-related Aβ mutants exhibited faster rates of aggregation. They also observed that Th-T fluorescence profiles of these FAD-related Aβ mutants displayed shorter times of lag phase with higher intensities of Th-T fluorescence and higher amounts of fibrils as compared to wild-type Aβ40, however, not all FAD-related Aβ mutants displayed the same patterns. Several FAD-related Aβ mutants showed a lower intensity of Th-T fluorescence with a higher amount of fibrils. This phenomenon can be explained by the binding ability of Th-T with Aβ aggregates or fibrils, since Th-T would bind to aggregates or fibrils of different structural conformations with distinct binding abilities, resulting in different fluorescence intensities. Hatami et al. reported that the intensity of Th-T fluorescence is not correlated with the amyloid fibril content. It can also be applied to explain our data shown in Figures 7 and 8 in which the Th-T fluorescence intensity of Aβ40(L17A/F19A/E22Q) after 45 h was higher than that of Aβ40(E22Q) and the TEM images showed a smaller amount of aggregates and/or fibrils of Aβ40(L17A/F19A/E22Q). These observations also suggested that the fibril conformation of Aβ40(L17A/F19A/E22Q) should be different from that of Aβ40(E22Q).

Many studies have reported that the FAD-related mutations, Dutch-type and Arctic-type mutations, both of which are located at position 22 within the Aβ sequence, would result in an increase of the aggregation rate of Aβ [11,12,37,38]. However, the underlying mechanisms by which these two FAD-related mutations accelerate the aggregation process of Aβ remain elusive. In general, the aggregation process of Aβ would involve conformational changes and self-association which are closely related to the intrinsic structural propensity, the intramolecular interactions within the Aβ molecule, and intermolecular interactions between Aβ molecules. Thus, any factor which varies these properties would alter its aggregation behavior as we discussed in the previous paper [30,31]. In the previous study, we investigated the mechanism of why Arctic-type mutation accelerates Aβ aggregation from a structural point of view and proposed that Arctic-type mutation would reduce the α-helical propensity of the α/β-discordant region of Aβ, resulting in an acceleration of Aβ aggregation [30]. However, it remains unclear whether or not Arctic-type mutation would enhance or reduce the intramolecular and/or intermolecular interactions of Aβ, since it is difficult to measure these interactions. In this study, we applied the same approach to investigate the underlying mechanism of how Dutch-type mutation promotes Aβ aggregation. Our data indicated that Dutch-type mutation, unlike Arctic-type mutation, has no significance on the structural propensity of Aβ. According to our data, the structural propensity of Dutch-type Aβ<sup>40</sup> and its interaction with SDS micelle are almost the same as those of wild-type Aβ40. Thus, we speculated that Dutch-type mutation might alter the intramolecular and/or intermolecular interactions of Aβ, leading to an increase of the aggregation rate of Aβ. This is a very likely inference, even though these effects were not directly observed. A single mutation at the same position (position 22) within the Aβ sequence with a different amino-acid would result in a distinct mechanism by which it promotes Aβ aggregation. This might be the reason why different FAD-related mutations within the Aβ sequence displayed different clinical characteristics, such as cerebral amyloid angiopathy (CAA) for Dutch-type Aβ40.

For L17A/F19A mutation, our data suggested that one of the factors in determining its inhibition of the aggregation of Aβ40(E22Q) is through increasing the α-helical propensity of the α/β-discordant region of Aβ40(E22Q). This effect was also observed on wild-type Aβ<sup>40</sup> and Aβ40(E22G) [21,29]. Whether the L17A/F19A mutation could inhibit the aggregation of other FAD-related Aβ mutants through the same effect which is exerted on wild-type Aβ40, Aβ40(E22Q) and Aβ40(E22G) remains to be investigated. The possibility that the intramolecular and/or intermolecular interactions of Aβ would be altered by the L17A/F19A mutation cannot be ruled out. We characterized the effects of L17A/F19A mutation on the structural propensity and aggregation kinetics of wild-type, Arctic-type and Dutch-type Aβ40, however, the effects of L17A/F19A mutation on the structural propensity and aggregation kinetics of the more amyloidogenic Aβ<sup>42</sup> and its FAD-related mutants remain unclear. Since the fibril structures of Aβ<sup>40</sup> [25,26,28,39] and Aβ<sup>42</sup> [40–45] have been solved at the atomic resolution, the intramolecular interactions within Aβ molecule and intermolecular interactions between Aβ molecules can be grasped to some extent based on this structural information. The intramolecular interactions within Aβ<sup>40</sup> were located at K16-D23 and G29-M35 segments, which correspond to the α/β-discordant region and c-terminal α-helix, respectively. According this structural information, L17A/F19A mutation would

disrupt the intramolecular interaction within Aβ40. It can be seen from the fibril structure of Aβ<sup>42</sup> that residues Ile41 and Ala42 are involved in the intramolecular and intermolecular interactions, suggesting that these two residues would affect the aggregation kinetics of Aβ. According to the Aβ<sup>42</sup> fibril structure, we may also speculate that the L17A/F19A mutation would also disrupt the intramolecular interaction within Aβ42, leading to an alternation of the aggregative propensity of Aβ42. Knowing the effects of the mutations within the Aβ sequence may help us in developing agents for inhibition of the aggregation of Aβ.

#### **4. Materials and Methods**

#### *4.1. Preparation of A*β *Peptides*

The protocols for production of Aβ peptides were the same as those described in the previous studies [29–31]. All peptide samples were dissolved in 70% TFE (trifluoroethanol) and then lyophilized. For NMR studies, peptides were dissolved in 0.25 mL 100 mM SDS-d25 (sodium dodecyl sulfate-d25) with 10% (*v*/*v*) D2O/H2O containing 10 mM phosphate buffer at pH 6.0. TSP (3-(trimethylsilyl)propionic-2,2,3,3,-d4 acid) was used for internal chemical shift standard. The sample solutions were put into the 5 mm Shigemi tubes (Shigemi Co., Allison Park, PA, USA) for NMR spectra recording.

#### *4.2. Nuclear Magnetic Resonance (NMR) Spectroscopy*

NMR experiments were performed at 296 K on the Bruker AVANCE-500, 600, or 800 spectrometer equipped with a 5-mm inverse triple resonance (1H/ 13C/ 15N), Z-axis gradient cyroprobe. NMR data were processed and analyzed using the TopSpin and AURELIA programs (Bruker BioSpin GmbH, Rheinstetten, Germany). Linear predictions were used in the indirectly detected dimensions to improve digital resolution. 1H chemical shifts were referenced to the 1H frequency of the methyl resonances of TSP at 0 ppm. The 15N and 13C chemical shifts were indirectly referenced using the following consensus ratios of the zero-point frequencies: 0.101329118 for 15N/ 1H and 0.251449530 for 13C/ 1H [46]. Backbone sequential assignments were accomplished using the three-dimensional spectra: HNCOCA, HNCO, HNCA, and CBCA(CO)NH [30,31].

#### *4.3. Circular Dichroism (CD) Spectroscopy*

Pretreated Aβ peptides (50 μM) were dissolved in 0.160 mL 100 mM SDS containing 5 mM phosphate buffer at pH 6.0. CD measurements were performed on an AVIV CD spectrometer (Aviv 410 spectropolarimeter, Aviv Biomedical, Inc., Lakewood, NJ USA) at 296 K [30,31]. The measurement was carried out three times.

#### *4.4. Thioflavin T (Th-T) Fluorescence Assay*

Pretreated Aβ peptides (30 μM) were incubated in aqueous solution (5 mM phosphate buffer, pH 7.2). The molar ratio of Aβ and thioflovin T (Th-T) (Sigma) was 1:1. Fluorescence signals were acquired (SpectraMax M5, Molecular Device, San Jose, CA, USA) every 30 min at 37 ◦C. The excitation and emission wavelengths of fluorescence were 450 nm and 482 nm, respectively [21,29].

#### *4.5. Transmission Electron Microscopy (TEM)*

TEM images of Aβ peptides were acquired using JEOL JEM-2100 EXII TEM (JEOL, Tokyo, Japan). Pretreated Aβ peptides (60 μM) were dissolved in 10 mM phosphate buffer, pH 7.0 and incubated at 37 ◦C for different time periods. Sample preparation of TEM followed the procedures as described [29,30].
