Further examination of peptides with well-folded antiparallel beta strands as inhibitors of amyloid formation from alpha-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert alpha-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied beta hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a D-Pro-L-Pro turn locus across strand termini. At a 2 : 1 peptide to alpha-synuclein ratio, cyclo-WW2 displays complete inhibition of beta-structure formation. Trp-bearing antiparallel beta-sheets held together by a disulphide bond are also potent inhibitors. N-15 HSQC spectra of alpha-synuclein provided new mechanistic details. The time course of N-15 HSQC spectral changes observed during beta-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in N-15 HSQC spectra of alpha-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41-T54 span of alpha-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor.
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