Substituted Aβ42 peptides produced six different classes of oligomer
distributions (Fig. 2, Aβ42 panel, colored arrows), compared with two in
the Aβ40 case. The largest class (red) was characterized by decreased
amounts of pentamers and hexamers. The orange class (D-[H14,Q15] and
in addition to displaying exceptionally faint pentamer and hexamer
bands, produced a diffuse band with an
most consistent with trimer, but with substantial areas of this band
migrating at significantly lower Mr. These same
characteristics were observed with the dimer band. D-[D7, S8]
(purple arrow) was unique in that it showed increased intensity of
tetramer, pentamer, and hexamer. The D-[I31,I32] peptide also was
notable because it displayed the lowest amount of monomer relative to
the other bands in the lane. D-[S26,N27] also was unique in that
decreases in hexamer and trimer band intensities were observed in
concert with increased tetramer intensity (green arrow). D-[L34,M35]
and D-[M35,V36] peptides displayed exceptionally intense pentamer
and hexamer bands, but less intense trimer bands (blue arrows).
D-[V39,V40] and D-[I41,A42] showed exceptionally intense dimer
and trimer bands, but tetramers were difficult to detect (black arrows).
To determine the effects of di-D-amino acid substitutions on the time
dependence of β-sheet formation, a proxy for Aβ fibril formation
33-35 , we monitored
T (ThT) fluorescence (Fig. 3). The average maximum intensity
(FUmax)
of Aβ40 was 200 fluorescence units (FU). Aβ40 variants D-[V12,H13],
D-[K16,L17], D-[V18,F19], D-[F20,A21], D-[E22,D23],
D-[N27,K28], D-[L34,M35], and D-[V39,V40] displayed
substantially
greater
(>2×) intensities.
D-[Glu3, Phe4], and D-[Asp7,Ser8] had very similar maximum
intensities, all of which were ≈2× that of wild type Aβ40. The
intensities of D-[Asp23,Val24] and
<1/10, respectively, than that of wild type Aβ40. The time at
which half maximal fluorescence intensity occurred
(t1/2) was ~220 h for Aβ40.
D-[D01,A02], D-[E03, F04], D-[R05,H06], D-[H14,Q15],
D-[V18,F19], D-[E22,D23], and D-[S26,N27] displayed
substantially shorter (<1/2×) half times (Fig. 3, Table 1).
D-[V12,H13], D-[N27,K28], and D-[A30,I31], and
D-[L34,M35] displayed longer half times, all of which were
(<2×). We also calculated the change in fluorescence per unit
time (dFU/dt) during the quasi-linear phases of growth for each peptide
(Table 1). Cross-correlation of the dFU/dt,
and FUmax determined by linear fitting to each of the
respective scatter plots revealed a correlation (R=0.73) between dFU/dt
and FUmax (data not shown). No correlations were
observed dFU/dt and t1/2 or between
FUmax and t1/2.
In experiments using di-D-amino acid substituted Aβ42 variants, the
range of maximum fluorescence intensities was ~50-350,
approximately an order of magnitude lower than that of the Aβ40
peptides. Aβ42 displayed a maximum fluorescence intensity of ≈130 FU,
thus the relative differences in intensity between the substituted
peptides and wild type Aβ42 often were much smaller than in the Aβ40
case. D-[M35,V36] and D-[H14,Q15] produced the highest
intensities, ≈3× that of wild type Aβ42. The intensities of
D-[I41,A42], D-[F20,A21], and D-[Ser26, Asn27] were ≦2× that
of wild type Aβ42. D-[V12,H13], D-[Asn27,Lys28], and D-[Ala31,
Ile32] had the lowest maximal intensities. The remaining peptides
fluoresced with intensities less than or equal to that of wild type
Aβ42. The t1/2 for WT Aβ42 was 90 h. With the exception
of D-[Phe20, Ala21], which had a t1/2 of 190 h, all
of the other substituted peptides displayed t1/2 values
of ~10 h. Many di-D-amino acid substitutions resulted in
somewhat slower emanation of β-sheet structure, a phenomenon also
observed in the Aβ40 system, as well as producing increased half times.
D-[Y10,E11], D-[H14,Q15], D-[S26,N27] and D-[A30,I31]
assembled the slowest, with half times that all were > 200
h. Only D-[E22,D23] had a half time that was substantially lower
than that of WT Aβ42 (68 vs. 90 h).