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.