Morphology of single D-amino acid substituted Aβ
To determine the morphologies of the assemblies present after
conformationally changes ceased (as assessed by CD), we used negative
stain transmission electron microscopy (EM) (Fig. 9). Aliquots were
removed from the same peptide assembly reactions studied by CD so that
direct comparisons were possible. No fibrils were observed in any
samples immediately after preparation, with the exception of Aβ42 D-F20
peptide . Aliquot removed at intermediate and end times all contained
fibrils.
End-stage fibrils of WT Aβ40 were long (>1 mm), had average
diameters of ~12 nm, and displayed a helical twist with
a periodicity of ~140 nm (Fig. 9, red arrows).
Interestingly, D-L17 peptides formed such structures at intermediate
times (50 h). D-L17 also displayed monofilar structures with average
diameters of ~6 nm as well as shorter filaments ranging
in length from 40-100 nm. End-stage D-N27 fibrils had diameters of
~10 nm, slightly narrower than those of WT Aβ40, and
lengths exceeding 1 mm. Short filaments of lengths 40-100 nm also
observed.
End-stage Aβ42 were long (>1 mm), had diameters of ≈10 nm,
and displayed a helical twist with a periodicity of
~140-250 nm (Fig. 9, Aβ42, red arrows). We observed two
predominant fibril morphologies with D-H14, the first was similar to
that of WT Aβ42 whereas the second was characterized by thinner fibrils
with average diameters of ~8 nm. These filaments were
highly twisted with a pitch of ~25 nm (black arrows).
These highly twisted filaments tended to be shorter (average length 450
nm) (Fig. 9, D-H14, inset) (inset)). Interestingly, D-F20 and D-A21
fibrils also displayed both types of morphologies as those observed in
D-H14, though for D-F20 they appeared as early as 12 h after the start
of incubation (the micrograph of D-F20 shown in Fig. 9 is the 12 h time
point). The fibrils observed for D-M35 were relatively straight and also
had an average diameter of ≈10 nm. They displayed much less twisting
than did fibrils formed by the other peptides. Distinct filaments of
~5 nm in diameter, and with lengths of 40- 600 nm, also
were observed in the D-M35 sample, as were occasional trifilar
structures (Fig. 9, D-M35, asterisk).