Oligomerization of mono-D-amino acid substituted Aβ
We next synthesized a series of mono-D-substituted peptides (13 for
Aβ40, 24 for Aβ42) (Fig. 4). These peptides were chosen based on the
results of the PICUP and ThT experiments, which revealed the dipeptide
regions of Aβ that most affected oligomerization and β-sheet formation.
For each of these dipeptide regions, we synthesized the two
corresponding mono-substituted peptides. We then studied oligomerization
behaviors using PICUP, SDS-PAGE, and silver staining. We included
non-cross-linked peptides in each case because prior studies had shown
that Aβ42, but not Aβ40, formed SDS-induced trimers (predominately) and
tetramers 10. We wanted to determine if and how
D-amino acid substitution might affect this process. Notwithstanding the
prior reported lack of SDS effects on Aβ40 oligomer formation during
SDS-PAGE, we included the non-cross-linked Aβ40 peptides as a control
and to determine if the prior results were reproducible.
Non-cross-linked Aβ40 displayed an intense monomer band, along with a
faint doublet band migrating at an Mr consistent
with that of dimer (Fig. 5a). This faint doublet also was observed in
the lanes of the D-H14, D-N27, D-I31, and D-I32 peptides, but was not
readily visible in other lanes. We also observed a band migrating below
the monomer band that may represent an intramolecularly cross-linked
monomer. This band was most apparent with the D-H14 and D-Q15 peptides
Cross-linked Aβ40 produced an oligomer distribution comprising
predominately dimers→tetramers, with less intense pentamer and hexamer
bands (Fig. 5, Aβ40, XL). D-N27 showed the greatest difference in
oligomer distribution compared to Aβ40, with smeared tetramer, pentamer,
and hexamer bands. D-H14 and D-Q15 displayed less intense tetramer and
pentamer bands, as well as a band at an Mr lower than
monomer, consistent with what we observed in Aβ40 D-[H14,Q15] (Fig.
5, XL). This band was present in D-26, D-N27, D-I31, and D-I32 albeit
less intense. Relatively small amounts of monomer were observed in
D-A21, D-S26, D-I31, and D-I32 peptides. Although the nominal amounts of
peptide loaded into each well were identical, variations in total band
intensity can occur. For this reason, we performed densitometry on each
lane and calculated the normalized intensities of bands (Fig. S?). These
intensities were consistent with the observations presented above.
Non-cross-linked Aβ42 produced prominent monomer and trimer bands, and a
sharp tetramer band of lower intensity. The trimer band had a
characteristic trapezoidal shape 10. The
substituted peptides displayed a variety of patterns, ranging from
indistinguishable from Aβ42
D-V24, D-S26, D-N27, and D-K28: black circles) to the presence of only
an intense monomer band (D-F20, D-I31, D-I32, D-M35, D-V39, D-V40, and
D-I41: red diamonds). Some peptides had relatively little trimer and
tetramer (D-D01, D-H14, D-E22, D-D23, and D-L34: blue squares), only
small amounts of trimer (D-A30 and D-A42: grey stars), or monomer and a
faint band migrating just above (D-Q15, D-A21, and D-V36: green
triangles) (Fig. 5c non-XL).
Cross-linking of Aβ42 produced a characteristic oligomer distribution
comprising predominately
dimers→heptamers
with a node at pentamer/hexamer 10. The
oligomer distribution of D-F20 differed most from wild type Aβ42 (as it
did in its un-cross-linked state), exhibiting only faint monomer,
relatively darker dimer, and faint trimer bands. Interestingly, this
variant was the most difficult to solubilize and its oligomer
distribution was unique amongst all the peptides studied. The extent of
the effects of D-amino acid substitution in the cross-linked states was
very similar to that in the non-cross-linked states (cf. Figs. 5c non-XL
vs. XL). If a particular peptide demonstrated essentially complete
elimination of oligomers in the non-cross-linked states, this same
peptide, when cross-linked, produced distributions in which the
predominant oligomer states were dimer and trimer (e.g., see the
substitutions from positions 30→42). Tetramer, pentamer, and hexamer
bands were seen in these samples, but they were faint relative to their
counterparts in WT Aβ42. Virtually no heptamer bands were visible in
these samples. Relative to the oligomer distribution produced by WT
Aβ42, the D-01, D-H14, and D-Q15 variants displayed more intense dimer
and trimer bands with less intense tetramer, pentamer, and hexamer.
Several oligomer distributions were devoid of a tetramer band (D-Q15 and
D-A21). Others had very faint monomer bands compared to Aβ42 (D-F20,
D-D23, D-M35, D-V36, and D-V40). The D-S26, D-N27, and D-K28 peptides
produced distributions that were quite similar to that of WT Aβ42.
Densitometric analyses of the normalized band intensities were
consistent with these observations (Fig. ?????????). Fig. ??????
presents histograms of representative examples for each of the five
classes is shown in Fig. 5d.