*Article* **Conformational Characterization of Native and L17A**/**F19A-Substituted Dutch-Type** β**-Amyloid Peptides**

**Kai-Cyuan He 1,2, Yi-Ru Chen 3,**†**, Chu-Ting Liang 1,4, Shi-Jie Huang 2, Chung-Ying Tzeng 4, Chi-Fon Chang 5, Shing-Jong Huang 6, Hsien-Bin Huang 7,\* and Ta-Hsien Lin 1,2,4,\***


Received: 29 February 2020; Accepted: 6 April 2020; Published: 7 April 2020

**Abstract:** Some mutations which occur in the α/β-discordant region (resides 15 to 23) of β-amyloid peptide (Aβ) lead to familial Alzheimer's disease (FAD). In vitro studies have shown that these genetic mutations could accelerate Aβ aggregation. We recently showed that mutations in this region could alter the structural propensity, resulting in a different aggregative propensity of Aβ. Whether these genetic mutations display similar effects remains largely unknown. Here, we characterized the structural propensity and aggregation kinetics of Dutch-type Aβ<sup>40</sup> (Aβ40(E22Q)) and its L17A/F19A-substituted mutant (Aβ40(L17A/F19A/E22Q)) using circular dichroism spectroscopy, nuclear magnetic spectroscopy, and thioflavin T fluorescence assay. In comparison with wild-type Aβ40, we found that Dutch-type mutation, unlike Artic-typemutation (E22G), does not reduce theα-helical propensity of theα/β-discordant region in sodium dodecyl sulfate micellar solution. Moreover, we found that Aβ40(L17A/F19A/E22Q) displays a higher α-helical propensity of the α/β-discordant region and a slower aggregation rate than Aβ40(E22Q), suggesting that the inhibition of aggregation might be via increasing the α-helical propensity of the α/β-discordant region, similar to that observed in wild-type and Artic-type Aβ40. Taken together, Dutch-type and Artic-type mutations adopt different mechanisms to promote Aβ aggregation, however, the L17A/F19A mutation could increase the α-helical propensities of both Dutch-type and Artic-type Aβ<sup>40</sup> and inhibit their aggregation.

**Keywords:** NMR; CD; Aβ; β-amyloid peptide; α/β-discordant; Dutch-type mutation; E22Q; familial Alzheimer's disease; FAD

#### **1. Introduction**

On the basis of the amyloid cascade hypothesis [1,2], aggregation of β-amyloid peptide (Aβ) is a crucial factor for the neuronal damage that leads to Alzheimer's disease (AD). The clinical hallmarks of AD are neurofibrillary tangles and senile plaques within AD patients' brains. The major components of these two hallmarks are tau protein and Aβ, respectively. Aβ, about 38–42 residues in length, is a derivative from sequentially enzymatic processing of transmembrane protein, called β-amyloid precursor protein (βAPP). It has been reported that increased Aβ production resulting from mutations in the processing enzymes of βAPP (such as β- and γ-secretase) [3] or βAPP mutations close to the cutting site of the processing enzymes [4,5] would cause family Alzheimer's disease (FAD). Point mutations within the Aβ region of βAPP have also been shown to cause family Alzheimer's disease (FAD), such as mutations occurring at A21 [6], E22 [7,8], and D23 [9,10] of Aβ. Several studies have shown that E22G (Arctic-type mutation), E22Q (Dutch-type mutation), and D23N (Iowa-type mutation) mutations would alter the aggregation behavior [11] and structure property [12–17] of Aβ.

Structures of wild-type Aβ in different environments have been reported. They adopted a mainly random coil conformation [18] or a short α-helical structure in aqueous solution [19]. In SDS micellar solution, two short α-helices were contained [20–22]. In the presence of large unilamellar vesicles (zwitterionic lipid bilayers), a partially folded structure was shown [23]. In vitro experiments have shown that Aβ would aggregate into fibrils whose secondary structure was mainly β-sheets [24–26]. Similar β-sheet conformations were also observed for the Aβ fibrils purified from AD brain tissue [27,28]. These findings suggested that Aβ would undergo conformational transitions from random coil or α-helix conformation into β-sheet structure during the process of aggregation. However, the detailed mechanism of the aggregation process of Aβ remains unclear. The aggregative propensity of Aβ is linked to its structural conversion tendency which depends on its intrinsic structural propensity and the local environments where it exists.

Previously, we reported that mutations located in the α/β-discordant region (resides 15 to 23) of Aβ (E22G and L17A/F19A mutations) could either reduce or augment α-helical propensity of Aβ, leading to either an increase or a decrease of the rates of structural transition and fibril formation of Aβ [21,29–31]. The results of these studies support the view that the α-helical and aggregative propensities of Aβ tend to be inversely correlated. It remains uncertain whether other FAD-related mutations located in the Aβ sequence would promote Aβ aggregation by reducing the α-helical propensity of Aβ or not. We have been focusing on investigating the effects of FAD-related mutations in the α/β-discordant region of Aβ on the structural propensity of Aβ. The effect of Arctic-type mutation (E22G) on the structural propensity of Aβ has been reported [16,30], however, the effects of other FAD-related mutations on the structural propensity of Aβ remain largely unknown. In the present study, we applied nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies to characterize the structural conformation of Dutch-type Aβ<sup>40</sup> (Aβ40(E22Q) in SDS micellar solution. Moreover, the effects of Ala replacements at L17 and F19, which have been shown to increase the α-helical propensity and decrease the rate of aggregation of wild-type Aβ<sup>40</sup> and Arctic-type Aβ<sup>40</sup> (Aβ40(E22G)), on the structure and aggregation kinetics of Dutch-type Aβ40, were also characterized. Our data suggested that the structural conformation of Aβ40(E22Q) in SDS micellar solution is very similar to that of wild-type Aβ40. There is only a slight difference between these two structures. However, there is a more significant difference in α-helical propensity between Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q). These results are discussed in terms of the relation between the structural and aggregative propensities of Aβ mutants.

#### **2. Results**

### *2.1. Comparison of the Secondary Structures of Wild-Type A*β*<sup>40</sup> and A*β*40(E22Q)*

In our recent study, we reported the effect of Arctic-type mutation (E22G) on the structure of Aβ in SDS micellar solution. To gain more insight into the effect of FAD-related mutation at position 22 on the structure of Aβ, we characterized the structure of Aβ40(E22Q) in the present study. Aβ40(E22Q) had been found in FAD patients with severe cerebral amyloid angiopathy (CAA). To examine the effect of E22Q mutation on the structure of Aβ, we first analyzed the secondary structures of wild-type Aβ<sup>40</sup> and Aβ40(E22Q) in SDS micellar solution using circular dichroism (CD) spectroscopy. It can be seen from Figure 1 that the CD spectrum of wild-type Aβ<sup>40</sup> shows a band with positive ellipticity at around 192 nm and two bands with negative ellipticity at 207 nm and 221 nm which are CD spectral characteristics of α-helix, suggesting that the secondary structure content of wild-type Aβ<sup>40</sup> in micellar solution is mainly α-helix. The result is consistent with that obtained in the previous studies [30,31]. The CD spectrum of Aβ40(E22Q) displays a similar spectral pattern to that of wild-type Aβ<sup>40</sup> with a more positive ellipticity at around 192 nm and a slightly more negative ellipticity at 207 nm and at 221 nm, suggesting that Aβ40(E22Q) adopts mainly α-helical conformation as well, and the α-helix content of Aβ40(E22Q) might be slightly higher than that of wild-type Aβ40.

**Figure 1.** Overlay of CD spectra of wild-type Aβ<sup>40</sup> (**black**), Aβ40(E22Q) (**red**) and Aβ40(L17A/F19A/E22Q) (**blue**) in 100 mM SDS micellar solution.

We further applied NMR spectroscopy to characterize the secondary structure of Aβ40(E22Q) in SDS micellar solution. In order to derive the secondary structure from the backbone atom chemical shifts, we first accomplished the sequential backbone assignment of Aβ40(E22Q). Figure 2A shows the two-dimensional 1H-15N-HSQC spectrum of 15N-labeled Aβ40(E22Q) in SDS micellar solution. The result of residue assignment is shown in the figure. By comparison of the two-dimensional 1H-15N-HSQC spectrum of Aβ40(E22Q) with that of wild-type Aβ40, we obtained the effect of E22Q mutation on the two-dimensional 1H-15N-HSQC spectrum of wild-type Aβ40. Figure 2B showed the superimposed two-dimensional 1H-15N-HSQC spectra of wild-type and Dutch-type Aβ40. It is evident that these two spectra look almost the same except for some amide proton and nitrogen cross-peaks which displayed chemical shift changes as a result of E22Q mutation. According to the previously assigned two-dimensional 1H-15N-HSQC spectrum of wild-type Aβ<sup>40</sup> [30], some cross-peaks which displayed relatively significant chemical shift changes on account of E22Q mutation were readily assigned to L17, V18, F20, A21, and D23 (excluding E22). In general, there are three major factors which contribute to the observed chemical shift perturbations of nitrogen (15N) and amide proton (1HN), including the sequence effect caused by E22Q mutation, the conformational change induced by E22Q mutation, and the interaction with SDS micelle altered by E22Q mutation. Further analysis revealed that the chemical shift perturbations are very small (less than 0.05) as shown in Figure 2C, suggesting that the effects of E22Q mutation on these three factors which cause chemical shift perturbations are

very small. It can also be seen from Figure 2C that residues which displayed relatively significant chemical shift perturbations resulting from E22Q mutation were located in the α/β-discordant region (resides 15 to 23). This observation suggested that E22Q mutation might slightly affect the structural conformation of the α/β-discordant region of Aβ and/or the interaction of the α/β-discordant region of Aβ with SDS micelle.

In order to confirm the inference that the effect of E22Q mutation on the structural conformation is small, we used secondary chemical shifts of 13C<sup>α</sup> and 13C<sup>β</sup> which are mainly affected by the backbone conformation of the amino acid itself instead of any direct through-space interaction, slightly affected by the sequence [32], to estimate the secondary structure of Aβ40(E22Q) [33–35]. Figure 3A shows the comparison of 13C<sup>α</sup> secondary chemical shifts of wild-type Aβ<sup>40</sup> and Aβ40(E22Q). It is apparent that the 13C<sup>α</sup> secondary chemical shifts of wild-type Aβ<sup>40</sup> and Aβ40(E22Q) look almost the same except for a few residues in the α/β-discordant region which displayed slightly more positive 13C<sup>α</sup> secondary chemical shifts resulting from E22Q mutation. This result suggested that both wild-type Aβ<sup>40</sup> and Aβ40(E22Q) adopted two short α-helices from residues 15 to 26 and residues 28 to 34 [35] and a few residues in the α/β-discordant region might have adopted slightly higher α-helical propensities (α-helicity) [33] as a result of E22Q mutation. By taking the 13C<sup>β</sup> secondary chemical shift into account, we further analyzed the effect of the E22Q mutation on the secondary structure of wild-type Aβ40. The results are shown in Figure 3B. As expected, the differences between 13C<sup>α</sup> and 13C<sup>β</sup> secondary chemical shifts of wild-type Aβ<sup>40</sup> and Aβ40(E22Q) look almost the same. It can be seen from Figure 3B that a few residues in the α/β-discordant region also displayed slightly more positive values of Δδ13C<sup>α</sup> –Δδ13C<sup>β</sup> for Aβ40(E22Q) than for wild-type Aβ40. This observation suggested that the E22Q mutation might result in a slight increase in the α-helical propensities of a few residues in the α/β-discordant region as well [34]. These findings were consistent with those observed from CD spectroscopy. Since the slight differences in 13C<sup>α</sup> secondary chemical shifts (or Δδ13C<sup>α</sup>–Δδ13C<sup>β</sup>) between wild-type Aβ<sup>40</sup> and Aβ40(E22Q) are within the error limits of chemical shift measurements using three-dimensional NMR spectra, one may argue that these relatively small differences might be overinterpreted. These differences might merely come from sequence effect. At any rate, we may speculate that the effects of E22Q mutation on the secondary structure of Aβ and the interaction of Aβ with SDS micelle are insignificant. Even though it exists, it is very small according to our NMR and CD data.

**Figure 2.** *Cont*.

**Figure 2.** (**a**) Two-dimensional 1H-15N-HSQC spectrum of 15N-labeled Aβ40(E22Q) in 100 mM SDS micellar solution at 296 K; (**b**) Overlay of two-dimensional 1H-15N-HSQC spectra of 15N-labeled wild-type Aβ<sup>40</sup> (black) and Aβ40(E22Q) (red) in 100 mM SDS micellar solution at 296 K. Residues which display chemical shift perturbations were labeled; (**c**) Chemical shift perturbation plotted as a function of residue number. Chemical shift perturbation was calculated using the equation [(HNΔppm)<sup>2</sup> + (NΔppm/10)2] <sup>1</sup>/2, where HNΔppm and <sup>N</sup>Δppm were equal to 1HN and 15N chemical shift differences between wild-type Aβ<sup>40</sup> and Aβ40(E22Q), respectively [31].

**Figure 3.** (**a**) 13C<sup>α</sup> secondary chemical shifts of wild-type Aβ<sup>40</sup> (balck) and Aβ40(E22Q) (red) plotted as a function of residue. In principle, if the 13C<sup>α</sup> secondary chemical shift of an amino acid residue is greater than 0.7 ppm, its conformation would be α-helical [35]; (**b**) Differences between Δδ13C<sup>α</sup> ( 13C<sup>α</sup> secondary chemical shift) and Δδ13C<sup>β</sup> ( 13C<sup>β</sup> secondary chemical shift) of wild-type Aβ<sup>40</sup> (black) and Aβ40(E22Q) (red) plotted as a function of residue. Δδ13C<sup>α</sup> (or Δδ13C<sup>β</sup>) was defined as the difference between the observed 13C<sup>α</sup> (or 13C<sup>β</sup>) chemical shift of an amino acid residue and its 13Cα (or 13Cβ) chemical shift in a random coil conformation. If Δδ13C<sup>α</sup>–Δδ13C<sup>β</sup> for an amino acid residue is positive, its conformation would be α-helical. For a more detailed description of the relationship between the value of Δδ13C<sup>α</sup>–Δδ13C<sup>β</sup> and secondary structure of an amino acid residue please see the reference [34].
