We examined the effect of methionine oxidation of human recombinant K K-synuclein on its structural properties and propensity to fibrillate. Both oxidized and non-oxidized K K-synucleins were natively unfolded under conditions of neutral pH, with the oxidized protein being slightly more disordered. Both proteins adopted identical partially folded conformations under conditions of acidic pH. The fibrillation of K K-synuclein at neutral pH was completely inhibited by methionine oxidation. This inhibitory effect was eliminated at low pH. The addition of oxidized K K-synuclein to the unoxidized form led to a substantial inhibition of K K-synuclein fibrillation. ß

Amyloid ␤-protein (A␤) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). Recently, to elucidate the oligomerization pathway, we studied A␤ monomer folding and identified a decapeptide segment of A␤, 21 Ala-22 Glu-23 Asp-24 Val-25 Gly-26 Ser-27 Asn-28 Lys-29 Gly-30 Ala, within which turn formation appears to nucleate monomer folding. The turn is stabilized by hydrophobic interactions between Val-24 and Lys-28 and by longrange electrostatic interactions between Lys-28 and either Glu-22 or Asp-23. We hypothesized that turn destabilization might explain the effects of amino acid substitutions at Glu-22 and Asp-23 that cause familial forms of AD and cerebral amyloid angiopathy. To test this hypothesis, limited proteolysis, mass spectrometry, and solution-state NMR spectroscopy were used here to determine and compare the structure and stability of the A␤(21-30) turn within wild-type A␤ and seven clinically relevant homologues. In addition, we determined the relative differences in folding free energies (⌬⌬G f) among the mutant peptides. We observed that all of the disease-associated amino acid substitutions at Glu-22 or Asp-23 destabilized the turn and that the magnitude of the destabilization correlated with oligomerization propensity. The Ala21Gly (Flemish) substitution, outside the turn proper (Glu-22-Lys-28), displayed a stability similar to that of the wild-type peptide. The implications of these findings for understanding A␤ monomer folding and disease causation are discussed.A bundant evidence links the amyloid ␤-protein (A␤) with the neuropathogenesis of Alzheimer's disease (AD) (for recent reviews, see refs. 1 and 2). A␤ is a normal metabolite of the A␤ precursor (A␤PP), from which A␤ is produced by endoproteolysis (3). Two predominant forms of A␤ exist in vivo, A␤40 and A␤42, which are 40-and 42-aa in length, respectively (2, 4, 5). Recent experimental and clinical evidence suggests that the primary neurotoxins in AD are A␤ oligomers or protofibrils (1, 6-10). Understanding the folding and oligomerization of nascent A␤ monomers thus has become an especially important aspect of current strategies for understanding AD etiology and developing therapeutic agents.We have applied a multidisciplinary approach to the A␤ assembly problem. Initial studies used limited proteolysis coupled with mass spectrometry to determine whether monomeric A␤ possessed any stable or quasistable structure that could protect the peptide from proteolysis. Surprisingly, a 10-residue segment within both A␤40 and A␤42, Ala-21-Ala-30, was identified (11). The homologous decapeptide, A␤(21-30), displayed protease resistance identical to that of full-length A␤, suggesting that this region could organize monomer folding and thus be a folding nucleus. This suggestion was consistent with the observation that many folding nuclei studied in isolation are structurally stable (12-16). In fact, NMR studies of the A␤(21-30) peptide revealed a turn in the Val-24-Lys-28 region that was stabilized by hydrophobic interactions between...

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