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.