In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimer's disease.Many diseases share the common trait of peptide-protein misfolding that leads to oligomerization and, eventually, formation of plaques of β-sheet structure. Prominent among these are type 2 diabetes 1 , Parkinson's disease 2 and Alzheimer's disease 3,4 . Of these, Alzheimer's disease is the leading cause of late-life dementia and is the focus of this paper. An increasing body of evidence links oligomerization of a ubiquitous peptide, the amyloid-β [3][4][5][6] . For this reason, elucidation of pathways of oligomer formation may be critical for the identification of therapeutic targets.Many types of oligomeric amyloid-β assemblies have been described (for a review, see Lazo et al. 7 ). Recently, Bitan et al. [8][9][10] used photoinduced cross-linking of unmodified proteins (PICUP) to reveal that the 42-residue form of amyloid-β, Aβ42, formed (Aβ42) 5 and (Aβ42) 6 oligomers ('paranuclei') that could oligomerize to form structures of higher order. Aβ40 did not form paranuclei, but instead existed as a mixture of monomers, dimers, trimers and tetramers. Chen and Glabe 11 , in contrast, used fluorescence and gel electrophoresis to determine oligomer states of amyloid-β refolded from denaturing solutions. They observed only Aβ42 monomer and trimer bands, and no oligomers of Aβ40. Differences such as these may exist because of the diverse experimental systems used to monitor amyloid-β selfassociation. Also, it has been argued that, in addition to the intrinsic potential of amyloid-β to traverse different assembly pathways, flaws in experimental design may have misled researchers in their quest to elucidate fully the amyloid-β oligomerization process 12 . Hence there is significant uncertainty about amyloid-β oligomer states and their position and relevance to amyloid-β aggregation. Results and discussionWe used a different, more direct, method to probe the amyloid-β oligomerization process: ion mobility coupled with mass spectrometry [13][14][15] . Details are given in the Methods section.Here the results for Aβ40 are given as an example. The mass spectrum of Aβ40 is s...

Neurotoxic assemblies of the amyloid b-protein (Ab) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Ab assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Ab(1-40) and Ab(1-42). The results revealed a 10-residue, protease-resistant segment, Ala21-Ala30, in both peptides. Remarkably, the homologous decapeptide, Ab(21-30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24-Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a b-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24-Lys28 region of Ab nucleates the intramolecular folding of Ab monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Ab(1-40) and Ab(1-42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Ab(1-42) production. Our results suggest that therapeutic approaches targeting the Val24-Lys28 turn or the Ab(1-42)-specific C-terminal fold may hold promise.

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Folding and self-assembly of the 42-residue amyloid b-protein (Ab) are linked to Alzheimer's disease (AD). The 21-30 region of Ab,, is resistant to proteolysis and is believed to nucleate the folding of full-length Ab. The conformational space accessible to the Ab(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the ''native'' state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24-K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.

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...