Site-Specific Fluorescence Dynamics of α‑Synuclein Fibrils Using Time-Resolved Fluorescence Studies: Effect of Familial Parkinson’s Disease-Associated Mutations

α-Synuclein (α-Syn) aggregation is directly implicated in both the initiation and spreading of Parkinson’s Diseases (PD) pathogenesis. Although the familial PD-associated mutations (A53T, E46K, and A30P) are known to affect the aggregation kinetics of α-Syn <i>in vitro</i>, their structural differences in resultant fibrils are largely unknown. In this report we studied the site-specific dynamics of wild type (wt) α-Syn and its three PD mutant fibrils using time-resolved fluorescence intensity, anisotropy decay kinetics, and fluorescence quenching. Our data suggest that the N- and C-terminus are more flexible and exposed compared to the middle non-amyloid-β component (NAC) region of wt and PD mutant α-Syn fibrils. Yet the N-terminus showed great conformational heterogeneity compared to the C-terminus for all these proteins. 71 position of E46K showed more flexibility and solvent exposure compared to other α-Syns, whereas both E46K and A53T fibrils possess a more rigid C-terminus compared to wt and A30P. The present data suggest that wt and PD mutant fibrils possess large differences in flexibility and solvent exposure at different positions, which may contribute to their different pathogenicity in PD

The Newly Discovered Parkinson’s Disease Associated Finnish Mutation (A53E) Attenuates α‑Synuclein Aggregation and Membrane Binding

α-Synuclein (α-Syn) oligomerization and amyloid formation are associated with Parkinson’s disease (PD) pathogenesis. Studying familial α-Syn mutants associated with early onset PD has therapeutic importance. Here we report the aggregation kinetics and other biophysical properties of a newly discovered PD associated Finnish mutation (A53E). Our <i>in vitro</i> study demonstrated that A53E attenuated α-Syn aggregation and amyloid formation without altering the major secondary structure and initial oligomerization tendency. Further, A53E showed reduced membrane binding affinity compared to A53T and WT. The present study would help to delineate the role of A53E mutation in early onset PD pathogenesis

Biophysical characterization of isolated Mel and PP oligomers.

<p><b>(A)</b> CD spectroscopy of isolated oligomers of Mel and PP in the presence of heparin. Both oligomers showed helical conformation in CD. <b>(B)</b> ThT fluorescence of the isolated Mel and PP oligomers showing moderate ThT binding. <b>(C)</b> CR binding of the isolated Mel and PP oligomers. <b>(D)</b> EM images showing large globular oligomeric morphology of the isolated Mel and PP oligomers formed in the presence of heparin. Scale bar is 500 nm.</p

Oligomerization prediction of Mel and PP.

<p>The intrinsic oligomerization ability of Mel and PP peptide was calculated (at pH 5.5) using Zyggregator software. The positive values (in red) represent aggregation propensity of corresponding amino acid.</p

Structural characterization of Mel and PP.

<p><b>(A)</b> Structural model of Mel (red, PDB ID: 2MLT) and PP (blue, bovine PDB ID: 1BBA). <b>(B)</b> CD spectra of Mel and PP at day 0 (d0) and after 15 days (d15) in presence and absence of heparin. After the addition of heparin and subsequent incubation for two weeks, the secondary structure of PP remained mostly unchanged (helical). <b>(C)</b> FTIR spectra of two weeks incubated PP and Mel (in the absence and presence of heparin). Y-axis represents the absorbance (AU) and X-axis represents the wavenumber (cm^-1). Wavenumbers corresponding to the maximum absorbance are represented with arrow marks. Consistent with CD data, FTIR study also showed that in the presence of heparin, unstructured Mel transformed into helical conformation, whereas PP remained mostly helical both in presence and absence of heparin after incubation.</p

Morphological characterization of Mel and PP oligomers.

<p>EM and AFM analysis were performed to visualize the morphology of two weeks incubated Mel and PP (in the presence of heparin). EM (left panel) and AFM (middle panel) images showing oligomer formation in the presence of heparin. The right panel shows 3D AFM height images of oligomer. Scale bars for EM images are 500 nm. Height scales for AFM images are also shown.</p

Hydrodynamic radius of oligomers.

<p>Dynamic light scattering (DLS) was performed to obtain the hydrodynamic radius (Rh) of peptide samples incubated for two weeks in presence and absence of heparin. The Rh values of peptides incubated in the presence of heparin increased considerably.</p

Cytotoxic Helix-Rich Oligomer Formation by Melittin and Pancreatic Polypeptide

<div><p>Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson’s associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail.</p></div

Hydrophobic surface exposure of oligomers.

<p>Hydrophobic surface exposure in terms of NR binding by Mel and PP samples, incubated for two weeks in presence and absence of heparin. The data suggesting increased hydrophobic surface exposure during heparin-induced peptide oligomerization.</p

Biophysical characterization of Mel in presence of SDS and liposome.

<p><b>(A)</b> CD spectroscopy showing the helical conformation of Mel after immediate addition of SDS (2.5 mM) in Gly-NaOH buffer (20 mM, pH 9.2). (<b>B)</b> Mel showing immediate conversion to helical conformation after addition of liposomes. <b>(C)</b> AFM images of Mel (incubated in the presence of SDS at 37°C) showing large globular oligomers and some fibrillar species (shown in the inset). <b>(D)</b> ThT binding of Mel after 5 days of incubation in the presence of SDS.</p