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.