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...

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aβ, tau), α-synuclein, IAPP and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D) and amyotrophic lateral sclerosis (ALS) research, respectively for over many years.While SOD1 is a globular protein with a well-defined 3D structure, the Aβ, tau and α-synuclein proteins belong to the class of intrinsically disordered proteins (IDPs). IDPs are also known to play a critical role in many cellular functions such as signal transduction, cell growth, binding with DNA and RNA, and transcription, and are implicated in the development of cardiovascular problems and cancers 29 . The IDPs involved in neurodegenerative diseases have a few aggregation-prone regions and overall all IDPs have a low mean hydrophobicity and a high mean net charge 30 .IDPs are structurally flexible and lack stable secondary structures in aqueous solution. When isolated, they behave as polymers in a good solvent and their radii of gyration are well described by the Flory scaling law. 31 The insolubility and high self-assembly propensity of IDPs implicated in degenerative diseases have prevented high-resolution structural determination by solution nuclear magnetic resolution (NMR) and X-ray diffraction experiments. Local information at all aggregation steps can be, however, obtained by chemical shifts, residual coupling constants, and J-couplings from NMR, exchange hydrogen/deuterium (H/D) NMR, Raman spectroscopy; and secondary structure from fast Fourier infrared spectroscopy (FTIR) or circular dichroism (CD). Long-range tertiary contacts can be deduced from paramagnetic relaxation enhancement (PRE) NMR spectroscopy and single molecule Förster resonance energy transfer (sm-FRET), and short-range distance contacts can be extracted by cross linked residues determined by mass spectrometry (MS). Low-resolution 3D information of monomers and oligomers can be obtained by ion-mobility mass-spectrometry data (IM/MS) providing cross-collision sections, dynamic light scattering (DLS), pulse field gradient NMR spectroscopy and fluorescence correlation spectroscopy (FCS) providing hydrodynamics radius, small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), atomic force microscopy (AFM) and transmission electron microscopy (TEM) providing height features of the aggregates, as reported by some o...

The role of water in promoting the formation of protofilaments (the basic building blocks of amyloid fibrils) is investigated using fully atomic molecular dynamics simulations. Our model protofilament consists of two parallel β-sheets of Alzheimer Amyloid-β 16-22 peptides (Ac-K 16 -L 17 -V 18 -F 19 -F 20 -A 21 -E 22 -NH 2 ). Each sheet presents a distinct hydrophobic and hydrophilic face and together self-assemble to a stable protofilament with a core consisting of purely hydrophobic residues (L 17 ,F 19 ,A 21 ), with the two charged residues (K 16 , E 22 ) pointing to the solvent. Our simulations reveal a subtle interplay between a water mediated assembly and one driven by favorable energetic interactions between specific residues forming the interior of the protofilament. A dewetting transition, in which water expulsion precedes hydrophobic collapse, is observed for some, but not all molecular dynamics trajectories. In the trajectories in which no dewetting is observed, water expulsion and hydrophobic collapse occur simultaneously, with protofilament assembly driven by direct interactions between the hydrophobic side chains of the peptides (particularly between F-F residues). For those same trajectories, a small increase in the temperature of the simulation (on the order of 20 K) or a modest reduction in the peptide-water van der Waals attraction (on the order of 10%) is sufficient to induce a dewetting transition, suggesting that the existence of a dewetting transition in simulation might be sensitive to the details of the force field parametrization.