Structured Regions of α-Synuclein Fibrils Include the Early-Onset Parkinson's Disease Mutation Sites

Abstract

α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinson’s disease (PD). Here, we use results from an extensive investigation employing solid-state NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD—A30P, E46K and A53T—are located in structured regions. We find that E46K and A53T mutations, located in rigid β-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.

Mutant Protein A30P α-Synuclein Adopts Wild-type Fibril Structure, Despite Slower Fibrillation Kinetics

“…These fibrils were then used to seed future uniformly 13 C, 15 N-labeled A30P AS fibrils. After the allotted time, fibril solutions were washed, dried, packed into 3.2-mm standard or thin wall rotors (Varian, Fort Collins, CO), and rehydrated with 36% (m/v) water according to previously described protocols (22 (27) mixing for 13 C homonuclear polarization transfer. All experiments were acquired under 13.3-kHz MAS and at a cooling gas temperature of 10°C with 90 standard cubic feet per hour flow, resulting in an actual sample temperature of 18 Ϯ 5°C.…”

“…Gly, Ala, Thr, Ser, Ile, and Pro) are distinctively identifiable by their unique chemical shift patterns and are highly sensitive to secondary structure (36 -40). Therefore, to evaluate slight variations between one fibril batch to the next, 13 C-13 C two-dimensional spectra with 50-ms DARR (27) mixing were acquired of three different batches of A30P AS fibrils, prepared as described previously for uniformly 13 C, 15 N-labeled WT AS fibrils (20,22). The linear regression analysis of two individual batches showed R 2 values of 0.996, 0.999, and 0.999 for 13 CЈ, 13 CA and 13 CB, respectively (Fig.…”

“…Once these cross-polarization transfers have occurred, the resulting polarization on the 13 CЈ nucleus is transferred through space to the side chain 13 C nuclei using the DARR mixing scheme. Using these three multidimensional experiments, we conducted chemical shift assignments of sequential residues using well established techniques for making de novo site-specific chemical shift assignments (22,(43)(44)(45)(46)(47)(48)(49). Fig.…”

Pressure effects on α-synuclein amyloid fibrils: An experimental investigation on their dissociation and reversible nature

“…More recently, it has been revealed by solid state NMR that fibrils obtained from A53T aSN in vitro show some structural variations in the core with respect to fibrils of the WT protein [14,15], while the same effect was not observed for A30P aSN fibrils [16]. Moreover, the two pathogenic mutants show an accelerated aggregation kinetics in vitro [17] and differences in flexibility and solvent exposure of some amino acids [18].…”

Low-threshold stimulated emission from lysozyme amyloid fibrils doped with a blue laser dye

“…1 Their distinct and varied three-dimensional structures are composed of one or more long chains of amino acids connected by peptide bonds, typically forming a-helices and b-sheets, as well as looping and folded chains. 2 Distinguishing microstructures can be found in amyloid fibrils, the aggregated state proteins that are considered to be involved in serious neurodegenerative diseases such as Alzheimer's, 3 Parkinson's, 4 or Huntington's. 5 Generally, amyloid fibrils are composed of antiparallel b-sheets oriented perpendicular to the long-axis of the fibril, which can be as long as several micrometres but with a diameter of only some 8-10 nm.…”