11 research outputs found
Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.
No full textPMCID: PMC3591419This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils
Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR
No full textStructural Intermediates during α-Synuclein Fibrillogenesis on Phospholipid Vesicles
No full textα-Synuclein (AS) fibrils are the main protein component
of
Lewy bodies, the pathological hallmark of Parkinson’s disease
and other related disorders. AS forms helices that bind phospholipid
membranes with high affinity, but no atomic level data for AS aggregation
in the presence of lipids is yet available. Here, we present direct
evidence of a conversion from α-helical conformation to β-sheet
fibrils in the presence of anionic phospholipid vesicles and direct
conversion to β-sheet fibrils in their absence. We have trapped
intermediate states throughout the fibril formation pathways to examine
the structural changes using solid-state NMR spectroscopy and electron
microscopy. The comparison between mature AS fibrils formed in aqueous
buffer and those derived in the presence of anionic phospholipids
demonstrates no major changes in the overall fibril fold. However,
a site-specific comparison of these fibrillar states demonstrates
major perturbations in the N-terminal domain with
a partial disruption of the long β-strand located in the 40s
and small perturbations in residues located in the “non-β
amyloid component” (NAC) domain. Combining all these results,
we propose a model for AS fibrillogenesis in the presence of phospholipid
vesicles
TALOS+ predicted backbone dihedral angles ψ and φ as a function of residue number.
No full text<p>(A) E46K and (B) A53T AS fibrils. Error bars based on the 10 best TALOS+ database matches. Representation of the secondary structure for WT (black), E46K (blue) and A53T (red) AS fibrils based on TALOS+ analysis (β-strands, arrows; turn or loop curved lines; not predicted, dashed lines). WT TALOS+ results based on those from Comellas <i>et al</i>.</p
Structured Regions of α-Synuclein Fibrils Include the Early-Onset Parkinson's Disease Mutation Sites
No full textComparison of relative signal intensities between the WT, E46K and A53T.
No full text<p>Normalized peak heights from CANCO experiments as a function of residue number for (A) E46K, (B) A53T and (C) WT. Error bars correspond to the noise level. Representation of the secondary structure of WT AS fibrils based on TALOS+ analysis (β-strands, arrows; turn or loop curved lines; not predicted, dashed lines) from Comellas <i>et al</i>. Regions described to be essential for the fibril formation by Giasson <i>et al</i> are highlighted with a black outlined box. Grey boxes highlight the located of the WT β-strands.</p
Sequential backbone-walk used to obtain the chemical shift assignments of A53T.
No full text<p>Illustration of backbone connectivity through the NCACX (red), NCOCX (blue) and CAN(co)CX (black) spectra of residues A90-E83. In all cases the homonuclear mixing was achieved with 50 ms DARR.</p
Fibrils of mutant AS proteins prepared <i>in vitro</i> have a highly homogeneity and morphology similar to WT fibrils.
No full text<p>(A) AS fibrils formation of (blue circles) E46K, (red triangles) A53T and (black squares) WT monitored by the Thioflavin T fluorescence assay. Error bars were determined from seven replicates for each. Measurements were normalized to the highest fluorescence intensity obtained across all samples. (B) Comparison of the electron micrographs of (top) E46K, (middle) A53T and (bottom) WT AS fibrils. <sup>13</sup>C-<sup>13</sup>C 2D with 50 ms DARR mixing of (C) E46K and (D) A53T AS fibrils.</p
The E46K mutation causes major chemical shift perturbations throughout the AS fibril sequence.
No full text<p>(A) Expansions of <sup>13</sup>C-<sup>13</sup>C 2D spectral overlays (50 ms DARR mixing) of WT (black) and E46K (blue) AS fibril samples. (B) Plot of chemical shift perturbations between WT and E46K chemical shifts versus residue number. Residues labeled as (*) correspond to perturbations greater than 5 ppm (<sup>15</sup>N) or 3 ppm (<sup>13</sup>C). Residues labeled as (#) correspond to glycines. The mutation is indicated with (†). Error bars correspond to the chemical shift variations from one WT batch to another. WT chemical shift assignments were obtained from the BMRB #16939.</p
Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy
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