Site-Specific Fluorescence Polarization for Studying the Disaggregation of α-Synuclein Fibrils by Small Molecules

Abstract

Fibrillar aggregates of the protein α-synuclein (αS) are one of the hallmarks of Parkinson's disease. Here, we show that measuring the fluorescence polarization (FP) of labels at several sites on αS allows one to monitor changes in the local dynamics of the protein after binding to micelles or vesicles, and during fibril formation. Most significantly, these site-specific FP measurements provide insight into structural remodeling of αS fibrils by small molecules and have the potential for use in moderate-throughput screens to identify small molecules that could be used to treat Parkinson's disease.

Figure 1

Labeling α-Synuclein (αS) for Studies of Aggregation. Top: Protein sequence of αS. Green circles indicate positions for fluorophore attachment. Bottom Left: Cys mutants of αS are purified using a C-terminal intein tag, treated with β-mercaptoethanol (β-ME) to cleave the intein, and labeled with fluorescein maleimide (Fam). Bottom Right: Monomers of αS can form helices when bound to membranes. Disordered monomers can misfold and aggregate to form oligomers or fibrils. Aggregation experiments are performed with 1% labeled protein to avoid fluorophore/fluorophore interactions.

Figure 2

Fluorescence Polarization (FP) Reveals Local Dynamics in Proteins Bound to Micelles or Vesicles. Left: FP measurements of αS constructs labeled with Fam at the noted positions, bound to sodium dodecyl sulfate (SDS) micelles or large unilamellar vesicles (LUVs) composed of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). Middle Right: The SDS-bound structure of αS (PDB ID: 1XQ8) and a model of the LUV-bound αS structure, colored to match the sequence diagram in Figure 1, are shown for comparison to the FP data.^, Far Right: Images of Fam-labeled αS constructs bound to giant unilamellar vesicles (GUVs) composed of DOPS and Texas red-1,2-dihexadecanoyl-sn-glycero-3 phosphoethanolamine. Numbers indicate the position of the Fam label on αS constructs. Scale bar = 10 μm.

Figure 3

Monitoring Forward Aggregation and Validating Labeled Constructs. Top Left: Aggregation kinetics of fibrils made with WT αS or 1% αS-C^Fam₉ monitored by the ratio of Congo Red (CR) absorbance ratio (540 nm/480 nm) as well as changes in FP. Top Right: Transmission electron microscopy (TEM) images show that fibrils made with 1% αS-C^Fam₉ are morphologically similar to WT αS fibrils. Scale bar = 100 nm. Bottom: Gel analysis demonstrates that αS-C^Fam₉ is incorporated into fibrils stoichiometrically. Standards (Stnds) of 1% αS-C^Fam₉ loaded at 100, 50, 25, and 12.5 μM. Molecular Weight (MW) markers at 11, 17, 22, 25, 32, 46, and 58 kDa. The same battery of experiments were conducted on αS constructs labeled at Cys 24, 42, 62, 87, 114, 123, or 136. These data are shown in Supporting Information.

Figure 4

Monitoring Fibril Disaggregation by FP. A–D: Fibrils made with 1% Fam-labeled αS constructs (at positions 9, 24, 42, 62, 87, 114, 123, or 136) were incubated with solutions of buffer, dopamine (Dop), epigallocatechin gallate (EGCG), or nordihydroguiaretic acid (NDGA). FP measurements are normalized to aid comparison of kinetics. Raw FP values are reported in Supporting Information. E: After incubation for 3 h, the reactions were pelleted by centrifugation. A portion of the insoluble fraction was boiled in SDS and loaded onto a gel for quantification relative to buffer-treated fibrils, as described in Supporting Information. F–G: FP measurements of the resuspended insoluble and soluble fractions are shown. FP data for untreated fibrils and for monomer are shown for comparison. H–K: TEM images of WT αS fibrils, treated with the same disaggregation protocol, deposited on copper grids, and negatively stained with ammonium molybdate. Scale bar = 200 nm. Larger TEM images are shown in Supporting Information, Fig. S31 a–d.