Introduction
Alzheimer’s disease (AD) is the most common cause of late-life dementia 1 and recently has been identified as the 6th leading cause of death in the U.S 2. There thus is a compelling need for the development of approaches to prevent, ameliorate, or cure this tragic disorder. A predominant working hypothesis of disease causation posits that oligomeric forms of the amyloid β-protein (Aβ) are key neurotoxic agents 3 . If so, then therapeutic drug development requires appropriate targeting of these assemblies. A variety of targeting strategies have been executed, including those directed against the enzymes responsible for Aβ production (β-secretase and γ-secretase) 4 or immunogenic sites on monomeric, oligomeric, and fibrillar forms of Aβ 5 . Unfortunately, none have resulted in an FDA-approved therapeutic agent 6.
Aβ exists in humans predominantly in two forms, Aβ40 and Aβ42, that are 40 or 42 amino acid residues in length, respectively 7 . Aβ42 appears to be the most disease-relevant peptide 8, 9 10. Mutations in the gene encoding the amyloid precursor protein (APP), from which Aβ42 is produced, lead to single amino acid substitutions linked to familial forms of AD and cerebral amyloid angiopathy. Most mutations result in single amino acid substitutions 11-23 . One results in the deletion of Gly22 11 . In vitro studies of the conformational dynamics and assembly of Aβ peptides containing these substitutions show that they facilitate folding, oligomerization, or fibril formation by the initially disordered Aβ monomer [insert Reference]. However, familial AD and CAA are estimated to account for only 5-10% of all AD cases [insert Reference]. This means that in the majority of AD cases, Aβ-mediated neurotoxicity and plaque formation are caused by wild type Aβ.
We sought here to determine which amino acids in wild type Aβ40 and Aβ42 have the greatest effects on peptide folding and assembly. Prior approaches for answering this question often have relied on amino acid substitution strategies, which have been informative and useful. However, by definition, the substituted amino acids differ from the wild type amino acids in polarity, charge, hydrophobicity, or size of the amino acid side-chains. These differences per se may be responsible for any changes, or lack of changes, in peptide folding and assembly, as opposed to the changes indicating how the wild type amino acid may or may not mediate these processes. To avoid these interpretive difficulties, we employed a scanning D-amino acid substitution strategy. We did so because this approach does not perturb peptide backbone geometry, nor does it alter polarity, charge, hydrophobicity, or size of the amino acid side-chains of the substituted residues. The only structural alteration is the orientation of the side-chain relative to the peptide backbone 24-27 . This approach thus reveals amino acids whose side-chains are involved in inter-atomic interactions that are exquisitely sensitive to structural perturbation and thus may be of special importance in controlling Aβ assembly and toxicity. In much the same way that study of transition states in chemical and enzymatic reactions are critical for establishing a mechanistic understanding of such reactions, chiral inversions may enable the study of peptide folding trajectories rarely traversed by the wild type peptide but that are critical for the production of conformers associated with pathologic Aβ assembly 28, 29 (see Raskatov and Teplow 27 for a theoretical treatment of the scanning D-amino acid strategy). Our experimental design comprised initial studies of the effects of scanning di-D amino acid substitution on peptide oligomerization and fibril formation, followed by determination of the effects of single D-amino acid substitutions chosen based on the data obtained in the initial studies. Our results enabled the mapping of the effects of specific amino acids on different aspects of peptide assembly and the comparative analysis of the maps of Aβ40 and Aβ42.