Experimental strategy
We executed a scanning D-amino acid substitution strategy in two phases
that involved the synthesis and study of a total of 76 different D-amino
acid substituted Aβ peptides. Single D-amino acid replacements at each
position along amphipathic model peptides have shown that a single
replacement may not always be sufficient for a reliable determination of
a structural effect 25. Pairwise substitution
of adjacent amino acids by their corresponding D-amino acids produces a
more substantial structural change 24. For
these reasons, we first synthesized all possible di-D-amino
acid-substituted Aβ40 and Aβ42 isomers (Fig. 1). Positions containing
Gly residues were not included as members of any di-substituted sites.
Each of these initial 39 peptides (19 Aβ40 peptides and 20 Aβ42
peptides) was monitored for effects of the substitutions on
oligomerization and fibril formation. If effects were observed for a
particular di-D-amino acid substitution, each of the corresponding
singly substituted peptides then were synthesized and studied. For the
mono-substituted peptides, in addition to monitoring oligomerization and
fibril formation, CD was used to study time-dependent changes in
secondary structure and TEM was used to determine assembly morphology.