Fibril formation kinetics of Aβ
In Aβ40, we observed the most substantial differences in fibril formation kinetics between WT and D-K16, D-L17, D-S26, and D-N27 (Fig. 10). These data indicate that K16, L17, S26, and N27 play a key role in controlling the aggregation of Aβ. Studies support the hypothesis that the central hydrophobic cluster (CHC) – Aβ(17-21) – plays an important role in Aβ plaque competence and in the aggregation propensities of Aβ 44 . Substituting L17 with its D-enantiomer resulted in a substantial increase in aggregation kinetics compared to Aβ40, consistent with the increase observed in D-[K16,L17] (Fig. 3a and Fig. 10). Here, we establish that L17 of the CHC in Aβ40 is an important residue in this important structural feature of Aβ. Furthermore, as aforementioned, the Aβ V24-K28 turn-region is thought to be important in initiating Aβ assembly processes at the monomer level 41 . D-amino acid substitutions at S26 and N27 substantially reduced the maximum ThT intensity of Aβ40 compared to WT. Since the D-amino acid substitutions at these sites reduced aggregation kinetics, S26 and N27 may be important residues controlling the aggregation propensity of Aβ40 as mutagenesis at these sites likely destabilized the turn in such a way that decreased the propensity of Aβ40 to aggregate. These data are consistent with previous studies done with the turn region that identified G25-K28 as important sites forming a β-turn (Lazo et al., 2005). The results from our study further establish that S26 and N27 are indeed important in nucleating monomer folding.
We also observed substantial differences in the fibril formation kinetics between Aβ42 and several of its single D-amino acid substituted variants (Fig. 5b and Fig. 10). These included Aβ42 D-H14, D-F20, D-A21, and D-M35 (Fig. 10). Similar to our studies of Aβ40 and its D-amino acid substituted variants, substitutions at residues in the CHC of Aβ42 altered the propensity of Aβ to form aggregates measured using ThT. Specifically, residues at the C-terminus of the CHC – F20 and A21 – displayed drastic changes in fibril formation kinetics compared to Aβ42. While a D-amino acid substitution at F20 increased fibril formation propensity, a D-amino acid substitution at A21 decreased it. These observations suggest that F20 of the CHC is important for preventing the formation of too many aggregates while A21 is an important residue for mediating the rapid aggregation usually observed with Aβ42 (Fig. 10). We also observed that M35 might be a key residue controlling the fibril formation kinetics of Aβ42 (Fig. 10). Previous studies implicated the C-terminus in controlling fibril assembly in Aβ42 (Yang and Teplow, 2008), and our study suggests that this may be regulated by M35.
PICUP showed that Aβ42 D-F20 and D-M35 displayed oligomer distributions shifted toward lower order oligomers compared to WT, while their ThT intensities were the two highest. This indicates that D-F20 and D-M35 aggregated rapidly – fewer monomeric structures were available to form oligomers, explaining the lower-order oligomers observed with these variants (Fig. 4e and 5b).
The secondary structure of the variants that substantially altered both the oligomer distribution and fibril formation kinetics of Aβ42 were studied using CD (Fig. 8). Based on these data, H14, F20, A21, and M35 may play key roles in controlling the pathway of secondary structure formation and establishing stable secondary structures in Aβ42 (Fig. 8b-f and Fig. 10). In addition, F20, A21, and M35 may be crucial in determining the rate at which Aβ42 transitions to its final secondary structure (Fig. 8g and Fig. 10). H14, F20, A21, and M35, identified in this study as important for the transition from statistical coil to β-sheet, have previously been shown to be within regions forming β-strands in Aβ42. One of the most recent studies predicts Aβ(16-20), Aβ(26-31), Aβ(35-36), and Aβ(39-41) as locations of β-strands in Aβ fibrils 45. Other studies predicted locations that included more residues, including H14 and A21 45 . We identify H14, F20, A21 and M35 as playing important roles in both secondary structure formation and controlling the rate of transition into stable secondary structures (Fig. 8), providing specific residues within the β-strands that might be crucial in stabilizing the β-strands.
Morphological analysis by electron microscopy showed that each of the variants studied formed fibrils. It seems that the D-substituted peptides that formed ThT positive structures the earliest appeared to have more likelihood of a highly twisted fibrillar structure present in addition to the typical fibrillar structure with a helical pitch similar to WT. At t = 0 h, fibrils were observed for Aβ42 D-F20, which is consistent with results from CD analysis, showing that D-F20 already possessed β-sheet content at 0 h (Fig. 6c), and the low quantities of oligomers observed by PICUP (Fig. 5c).
In this study, we have identified specific residues in both Aβ40 and Aβ42 that may be responsible in controlling oligomerization, fibril formation kinetics, and secondary structure formation (Fig. 10). H14, F20, A21, and M35 in Aβ42 and L17 and N27 in Aβ40 – when substituted with their D-enantiomers – substantially altered all of the studied characteristics of Aβ folding and assembly (Fig. 10). These residues may be relevant to target with drugs designed for AD therapy. Furthermore, amino acids at the C-terminus, which has previously been implicated in controlling the oligomerization of Aβ42, might also serve as drug targets for Aβ assembly inhibitors. The detailed structure-assembly relationships established in these studies have the potential to provide specific targets for the design of therapeutic agents.