Genetically Encoded Aryl Alkyne for Raman Spectral Imaging of Intracellular α-Synuclein Fibrils

• Published in: Journal of molecular biology Vol. 435; no. 1; p. 167716
• Main Authors: Watson, Matthew D., Lee, Jennifer C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 15-01-2023
• Subjects: 4-ethynylphenylalanine; Alkynes - chemistry; alpha-Synuclein - chemistry; alpha-Synuclein - genetics; amyloid; Amyloid - chemistry; Cell Line, Tumor; Humans; Molecular Imaging; Parkinson’s disease; SH-SY5Y; Spectrum Analysis, Raman; unnatural amino acid; C≡C; 4-ethynylphenylalanine; SH-SY5Y; ThT; FCC; unnatural amino acid; FP; aaRS; JUNQ; amyloid; νmax; PD; α-syn; TDP-43; TEM; Parkinson’s disease; HPG; IPOD
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2022.167716

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•Detection of amyloids in cells requires unique spectral signatures.•The aryl alkyne FCC is a site-specific conformational probe of α-syn aggregation.•Cellular Raman mapping uniquely identifies internalized FCC-labeled α-syn fibrils.•Spectral analysis reveals differences in lipid and protein recruitment to fibrils.•Raman spectral imaging is a powerful approach to study amyloid structure in cells.
α-Synuclein (α-syn) is an intrinsically disordered protein involved in a group of diseases collectively termed synucleinopathies, characterized by the aggregation of α-syn to form insoluble, β-sheet-rich amyloid fibrils. Amyloid fibrils are thought to contribute to disease progression through cell-to-cell transmission, templating and propagating intracellular amyloid formation. Raman spectral imaging offers a direct characterization of protein secondary structure via the amide-I backbone vibration; however, specific detection of α-syn conformational changes against the background of other cellular components presents a challenge. Here, we demonstrate the ability to unambiguously identify cellularly internalized α-syn fibrils by coupling Raman spectral imaging with the use of a genetically encoded aryl alkyne, 4-ethynyl-l-phenylalanine (FCC), through amber codon suppression. The alkyne stretch (CC) of FCC provides a spectrally unique molecular vibration without interference from native biomolecules. Cellular uptake of FCC-α-syn fibrils formed in vitro was visualized in cultured human SH-SY5Y neuroblastoma cells by Raman spectral imaging. Fibrils appear as discrete cytosolic clusters of varying sizes, found often at the cellular periphery. Raman spectra of internalized fibrils exhibit frequency shifts and spectral narrowing relative to in vitro fibrils, highlighting the environmental sensitivity of the alkyne vibration. Interestingly, spectral analysis reveals variations in lipid and protein recruitment to these aggregates, and in some cases, secondary structural changes in the fibrils are observed. This work sets the groundwork for future Raman spectroscopic investigations using a similar approach of an evolved aminoacyl-tRNA synthetase/tRNA pair to incorporate FCC into endogenous amyloidogenic proteins to monitor their aggregation in cells.