Abstract
The aggregation and deposition of α-synuclein in Lewy bodies is associated with the progression of Parkinson's disease. Here, Mass Spectrometry (MS) is used in combination with Ion Mobility (IM), chemical crosslinking and Electron Capture Dissociation (ECD) to probe transient structural elements of α-synuclein and its oligomers. Each of these reveals different aspects of the conformational heterogeneity of this 14 kDa protein. IM-MS analysis indicates that this protein is highly disordered, presenting in positive ionisation mode with a charge state range of 5 ≤z≤ 21 for the monomer, along with a collision cross section range of ∼1600 Å(2). Chemical crosslinking applied in conjunction with IM-MS captures solution phase conformational families enabling comparison with those exhibited in the gas phase. Crosslinking IM-MS identifies 3 distinct conformational families, Compact (∼1200 Å(2)), Extended (∼1500 Å(2)) and Unfolded (∼2350 Å(2)) which correlate with those observed in solution. ECD-Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry (ECD-FT-ICR MS) highlights the effect of pH on α-synuclein structure, identifying the conformational flexibility of the N and C termini as well as providing evidence for structure in the core and at times the C terminus. A hypothesis is proposed for the variability displayed in the structural rearrangement of α-synuclein following changes in solution pH. Following a 120 h aggregation time course, we observe an increase in the ratio of dimer to monomer, but no gross conformational changes in either, beyond the significant variations that are observed day-to-day from this conformationally dynamic protein.
Highlights
K0 1⁄4 3ze 16N2π 0:5 1 μkBT Ω ð1ÞThe aggregation of and the pre-fibrillar aggregates formed by β-2-microglobulin has been studied using Ion Mobility (IM)-Mass Spectrometry (MS)3070 | Analyst, 2015, 140, 3070–3081Paper approaches by the Vachet,[4] Radford[5,6] and Ashcroft groups.[7,8,9,10] Bowers and co-workers have extensively studied amyloid-β11–16 with contributions from Robinson.[17]
The aggregation of and the pre-fibrillar aggregates formed by β-2-microglobulin has been studied using Ion Mobility Mass Spectrometry (IM-MS)
Parkinsons Disease (PD) is one of these diseases and the protein involved is α-synuclein, a major constituent of the characteristic Lewy bodies, where it presents in an organised fibrillar state.[28,29] α-synuclein is a member of a subset of proteins known as, intrinsically disordered proteins (IDPs), defined as lacking tertiary fold observable on the timescale of an NMR experiment. α-synuclein is a 140 amino acid protein encoded by a single gene, SNCA and in neurons it is localised predominantly in the presynaptic nerve terminals and nucleus.[30]
Summary
Paper approaches by the Vachet,[4] Radford[5,6] and Ashcroft groups.[7,8,9,10] Bowers and co-workers have extensively studied amyloid-β11–16 with contributions from Robinson.[17]. The aggregation pathway has been described as containing multiple intermediate oligomer structures including spherical[36] and ringshaped[31,37] forms and is susceptible to pertubation, through changes to environmental factors. These factors include agitation, temperature and pH.[38] The transition from native conformation to these amyloid deposits is not a single step, nor a single pathway, and in order to understand the mechanisms at play it is critical to identify the species present at each step. We use transmission electron microscopy to determine fibril formation under the solution conditions employed in MS studies
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