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.