Aβ oligomer formation
Several experiments were executed to determine whether or not D-amino
acid substitutions in Aβ40 and Aβ42 altered peptide behavior.
Specifically, we sought to determine which substitutions would affect
oligomerization, fibril formation kinetics, secondary structure
formation, and morphology. From the PICUP and SDS-PAGE analysis of
single D-amino acid substituted Aβ, individual amino acids were
identified which alter oligomer formation in Aβ40 and in Aβ42 (Fig. 10).
In Aβ40, the oligomerization of D-S26 and D-N27 differed most notably
from WT when single D-amino acid substitutions were made (Fig. 10). S26
and N27 thus significantly affect Aβ40 oligomerization. These results
are consistent with previous in hydro and in silico
studies which postulated the importance of S26 in V24-K28 turn
formation, a structure proposed to be important for the intramolecular
nucleation of unfolded Aβ monomer to folded monomer
40, 41 . Other D-amino substituted variants such
as D-H14, D-F20, D-A21, D-I31, and D-I32 also affected peptide
oligomerization but in a more subtle way (Fig. 10). D-H14, D-F20, D-I31,
and D-I32 thus may play more minor roles in controlling the oligomer
formation of Aβ40. While we were able to identify single D-amino acid
substitutions that changed the oligomer distribution of Aβ40, some of
these single D-amino acid substituted variants did not differ in Aβ
oligomer distribution while their di-D-amino acid substitution
counterparts did, for Aβ40. We observed a smear of protein produced by
Aβ40 D-[K16,L17] (Fig. 2a) – a smear that was not observed with
Aβ40. However, the single D-K16 and D-L17 substitutions did not seem to
produce oligomer distributions different than that of WT (Fig. 4b). This
suggests that the interactions between amino acids and both K16 and L17
may be important in controlling the oligomerization of Aβ40, but also
that there may be redundancy in the function of K16 and L17. Another
interpretation would be that the D-amino acid substitutions in the
di-D-amino acid substituted variants may be acting synergistically to
alter the oligomer distribution of Aβ, explaining why changes in
oligomer distribution were not observed with K16 and L17 were
substituted individually.
While we observed several differences in the oligomer distribution of
Aβ40 when di-D-amino acid substitutions were made, even fewer single
D-amino acid substitutions altered the oligomer distributions of Aβ40.
For instance, D-[K16,L17] shifted the oligomer distribution of Aβ40
toward higher order oligomers (Fig. 2a). However, when K16 and L17 were
substituted with their D-enantiomers individually, we did not observe a
substantial change in the oligomer distribution compared to Aβ40 (Fig.
5b).
Meanwhile, fourteen individual amino acids seemed to affect Aβ42
oligomerization: D01, H14, Q15, F20, A21, D24, A30, I31, I32, L34, M35,
V39, V40, and I41 (Fig. 10). The D-amino acid substitutions at the
C-terminus exhibited the greatest effect on Aβ oligomer formation,
causing both the non-cross-linked and cross-linked Aβ42 variants at the
C-terminus to display oligomer distributions more like Aβ40, with the
absence of higher order oligomers (Fig. 10). These results are
consistent with reports suggesting that a turn in the C-terminus plays
an essential function in oligomerization and the assembly differences
between Aβ40 and Aβ42 32, 41-43. In this study,
we found that single amino acid substitutions of several amino acids
(e.g., I31, I32, L34, M35, V39, V40, and I41) at the C-terminus of Aβ42
altered oligomer formation, thereby identifying amino acids in the
C-terminus that control oligomerization. This suggests that the exact
orientations of each of these amino acid side chains are essential in
making Aβ42 behave as it does, and that altering any one of these can
reduce the formation of higher order oligomers. This also suggests that
the native sequence, with amino acids in their L-conformation, is
uniquely suited to form high order oligomers. Furthermore, we identified
distinct sets of amino acids that controlled the oligomer formation of
Aβ40 and Aβ42, indicating that different residues govern the assembly of
the two isoforms and confirming that the two isoforms form oligomers
through distinct pathways 10. The densitometry
analysis of the oligomer distribution for Aβ42 variants supports the
qualitative analysis (Fig. 5d). Importantly, the cross-linking results
are consistent with previous studies, where the C-terminus of Aβ was
established as an important site in controlling oligomerization
32, 41, 43 .