*2.2. Comparison of the Secondary Structures of A*β*40(E22Q) and A*β*40(L17A*/*F19A*/*E22Q)*

In our recent study, we showed that residues L17 and F19 of Aβ played an important role in the structural and aggregative propensities of wild-type Aβ<sup>40</sup> and Aβ40(E22G) [21,29,31]. To examine whether the effects of Ala replacements at L17 and F19 on the structure and aggregative property of Aβ40(E22Q) are similar to those observed for wild-type Aβ<sup>40</sup> and Aβ40(E22G) or not, we performed structural characterization and aggregation kinetic study on Aβ40(L17A/F19A/E22Q). Prior to the experimental structural characterization of Aβ40(L17A/F19A/E22Q), we applied propensity-based prediction to the analyzed effects of E22Q and L17A/F19A mutations on the structural propensity of the α/β-discordant region of wild-type Aβ<sup>40</sup> and Aβ40(E22Q), respectively [31,36]. The results obtained from in silico studies implied that wild-type Aβ<sup>40</sup> and Aβ40(E22Q) adopt the same structural propensity in their α/β-discordant region. Unlike the E22G mutation which would alter the structural propensity of D23 from α-helix to β-strand, the E22Q mutation has no effect on the structural propensity of wild-type Aβ40. It can also be seen that the L17A/F19A mutation would alter the structural propensities of residues 15 to 21 in the α/β-discordant region of Aβ40(E22Q) from β-strand to α-helix as shown in Figure 4. The same effect has also been observed on wild-type Aβ<sup>40</sup> and Aβ40(E22G) [31].


**Figure 4.** The predicted secondary structures of the α/β-discordant regions of wild-type Aβ<sup>40</sup> Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q). β-strands predicted with high and low probability were denoted by the symbols E and e, respectively. α-helical structures predicted with high and low probability were denoted by the symbols H and h, respectively [31,36].

We next applied CD spectroscopy to examine the effect of L17A/F19A mutation on the overall secondary structure of Dutch-type Aβ40. The CD spectra of Aβ40(L17A/F19A/E22Q) are shown in Figure 1. It is apparent that Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q) exhibited similar spectral patterns in their CD spectra, suggesting that the overall secondary structure of Aβ40(L17A/F19A/E22Q) is similar to that of Aβ40(E22Q). They both adopt mainly α-helical structures in SDS micellar solution. However, it can be seen from Figure 1 that Aβ40(L17A/F19A/E22Q) displayed more positive ellipticity at around 192 nm and more negative ellipticity at 207 nm and 221 nm than Aβ40(E22Q), suggesting that the L17A/F19A mutation would result in an increase of the α-helix content of Aβ40(E22Q). The difference between the CD spectra of Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q) is more significant than that between wild-type Aβ<sup>40</sup> and Aβ40(E22Q), indicating that the effect of L17A/F19A mutation on the overall secondary structure of Dutch-type Aβ<sup>40</sup> is more prominent than that of E22Q mutation on the overall secondary structure of wild-type Aβ40.

We also applied NMR spectroscopy to characterize the secondary structure of Aβ40(L17A/F19A/E22Q) in SDS micellar solution and used the same approach as that employed for analyzing the effect of E22Q mutation on the structural conformation of Aβ to analyze the effect of L17A/F19A mutation on the structural conformation of Dutch-type Aβ40. A two-dimensional 1H-15N-HSQC spectrum of 15N-labeled Aβ40(L17A/F19A/E22Q) in SDS micellar solution with the result of residue assignment is shown in Figure 5A. Figure 5B shows the comparison of the two-dimensional 1H-15N-HSQC spectra of Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q). It is quite obvious that many amide proton and nitrogen cross-peaks of Aβ40(E22Q) display significant chemical shift changes because of L17A/F19A mutation. Cross-peaks which display significant chemical shift changes are indicated in the figure. Calculations of chemical shift perturbations were also performed for further analysis of the effect of the L17A/F19A mutation on the chemical shifts of the amide proton and nitrogen cross-peaks of Aβ40(E22Q). The results are shown in Figure 5C. Residues which exhibited significant chemical shift perturbations (greater than 0.05) were readily identified as E11, H13-F20 (excluding L17 and F19), Q22, D23, and G25. These residues are mainly located in the α/β-discordant region of Aβ40(E22Q), suggesting that the increases of α-helical content observed

from CD spectra are mainly from the residues in the α/β-discordant region of Aβ40(L17A/F19A/E22Q). Similar effects have also been observed on wild-type Aβ<sup>40</sup> and Aβ40(E22G) [31]. This finding implied that the L17A/F19A mutation would affect the structural conformation of the α/β-discordant region of Aβ40(E22Q) and the interaction of the α/β-discordant region of Aβ40(E22Q) with SDS micelle.

The effect of the L17A/F19A mutation on the secondary structure of Aβ40(E22Q) was also analyzed in terms of the changes of secondary chemical shifts of 13C<sup>α</sup> and 13C<sup>β</sup>. Figure 6A,B shows the plots of 13C<sup>α</sup> secondary chemical shifts and the values of Δδ13C<sup>α</sup>–Δδ13C<sup>β</sup> of Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q) as a function of residue, respectively. It can be seen from Figure 6A,B that residues which displayed significant changes in the 13C<sup>α</sup> secondary chemical shifts and the values of Δδ13C<sup>α</sup>–Δδ13C<sup>β</sup> as a result of L17A/F19A mutation were mainly located in the α/β-discordant region of Aβ40(E22Q). Moreover, both the 13Cα secondary chemical shifts and the values of Δδ13C<sup>α</sup>–Δδ13C<sup>β</sup> for the residues in the α/β-discordant region are significantly more positive for Aβ40(L17A/F19A/E22Q) than for Aβ40(E22Q). These findings suggested that Aβ40(L17A/F19A/E22Q) adopted two short α-helices from residues 15 to 26 and residues 28 to 34, and residues 15–26 of Aβ40(L17A/F19A/E22Q) adopted higher α-helical propensities than those of Aβ40(E22Q). It has to be noted that changes of these secondary chemical shifts are primarily contributed by structural conformational changes induced by the L17A/F19A mutation. Alternation of interaction with SDS micelle would result in changes of these secondary chemical shifts as well. We cannot rule out the possibility that interaction of the α/β-discordant region of Aβ40(E22Q) with SDS micelle would be altered due to the L17A/F19A mutation. However, whether interaction with SDS is strong or not, its effect on the changes of these secondary chemical shifts is small.

**Figure 5.** *Cont*.

**Figure 5.** (**a**) Two-dimensional 1H-15N-HSQC spectrum of 15N-labeled Aβ40(L17A/F19A/E22Q) in 100 mM SDS micellar solution at 296 K; (**b**) Overlay of Two-dimensional 1H-15N-HSQC spectra of 15N-labeled Aβ40(E22Q) (red) and Aβ40(L17A/F19A/E22Q) (blue) 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> + ( <sup>N</sup>Δppm/10)2] <sup>1</sup>/2, where HNΔppm and <sup>N</sup>Δppm were equal to 1HN and 15N chemical shift differences between Aβ40(E22Q) and Aβ40(L17A/F19A/E22Q), respectively [31].

**Figure 6.** (**a**) 13Cα secondary chemical shifts of Aβ40(E22Q) (black) and Aβ40(L17A/F19A/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 Aβ40(E22Q) (black) and Aβ40(L17A/F19A/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 13C<sup>α</sup> (or 13C<sup>β</sup>) 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].
