Secondary structure of single D-amino acid substituted Aβ
To examine the effects of D-amino acid substitutions on secondary structure dynamics, we monitored the assembly of Aβ40 (D-L17 and D-N27) and Aβ42 (D-H14, D-F20, D-A21, D-M35) peptides using circular dichroism (CD) spectroscopy. These peptides were selected for study because the substitutions produced the largest effects on oligomerization and emanation of β-sheets. WT Aβ40 and its singly substituted D-L17 and D-N27 forms all initially displayed spectra consistent with primarily statistical coil structure (monotonic decrease in [θ] from 260 to ~200 nm, at which point an upward inflection occurs). All three peptides then underwent a time-dependent two-state (isodichroic point at ≈205 nm) transition to β-sheet structure. This transition occurred most rapidly (>2× that of WT) for the D-N27 peptide (Fig. 7e), an observation consistent with this peptide’s especially rapid rate of increase in ThT fluorescence and its very high final fluorescence intensity (Fig. 6a). Similarly, D-N27, the peptide showing the lowest rate (<1/7 that of WT) of ThT fluorescence change and the lowest final fluorescence intensity, displayed the slowest transition to β-sheet in the CD experiment.
Studies of the WT Aβ42 and its singly substituted D-H14, D-F20, D-A21, and D-M35 forms showed initial spectra consistent with statistical structure and subsequent coil→β-sheet transitions (Fig. 8, a-e). These transitions have been shown to involve a transitory α-helix-containing intermediate 36, the presence of which was indicated by spectra in WT, D-H14, D-A21, and D-F20 obtained at 102 h, 77 h, 363 h, and 267 h, respectively. These spectra displayed inflections at ≈208 and ≈219 nm, ≈209 and ≈220 nm, ≈210 and ≈220 nm, and and ≈215 nm, respectively. The wavelength deviations from the ≈208 and ≈222 nm inflections for pure helix 37 occurs because the conformers in our experiments are not completely α-helical. The relatively small magnitudes of the molar ellipticity values for D-M35 precluded us from confidently assigning α-helix structure to any of the spectra. When we determined the relative rates of conformational change of the five peptides, we observed that the curves describing the time dependence of [θ] for WT, D-H14, and D-F20 appeared biphasic. A rapid decreasing initial phase was followed by a slower, longer phase. D-A21 and D-M35 did not show biphasic behavior. WT and D-H14 transitioned the most rapidly and at approximately the same rate (Fig. 8g), as determined by linear fitting of the longer phases of the curves. Each of the other three peptides transitioned more slowly (~5×).
Spectra obtained when conformational changes stopped revealed differences among the peptides (Fig. 8f). WT and D-H14 had high β-sheet content, as indicated by minima at ≈215 nm. Asymmetry in the trough observed in D-H14 between 210 and 225 nm may represent some helical content, but variance in these data is too high to argue this confidently. However, this asymmetry is more obvious in the spectra of D-F20 and D-A21, suggesting that the final conformational states of the assemblies formed by these peptides have substantial β-sheet content but may also contain α-helix. D-M35 may have a similar final conformation.