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).