Raman Spectroscopic Studies of Amyloids

National Heart, Lung, And Blood Institute

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Abstract

Raman spectral imaging is a powerful technique that couples the chemical specificity of Raman spectroscopy with the spatial resolution of a microscope. Raman spectroscopy is commonly used to measure protein amide bands, which arise from coupled vibrational modes of the polypeptide backbone. The position and widths of the amide bands depend on the peptide-bond angles and hydrogen-bonding patterns, and therefore, inform on protein secondary structure as well as local environment. Conformations of alpha-helix, beta-sheet, or random coil exhibit characteristic peak maxima, making quantification of structural compositions possible. In the past reviewing period, we completed two projects. First, we investigated the possible link between liquid-liquid phase-separation (LLPS) of proteins and the disease-related process of amyloid fibril formation. In order to determine a mechanistic connection to amyloid formation, protein conformation state(s) inside droplets were evaluated as a function of aging time with spatial resolution with Raman spectral imaging. Here, the C-terminal domain (CTD) of TAR DNA-binding protein 43 (TDP-43), an amyotrophic lateral sclerosis-related protein known to both phase-separate and form amyloids, was studied. Using both bright-field and Raman spectral imaging, droplet maturation of TDP-43CTD was directly observed, offering detailed structural information with spatial context which would be otherwise obscured by bulk measurements. The earliest aggregation events occur within droplets as indicated by the development of beta-sheet structure and increased thioflavin-T emission. Additionally, a slower appearance of filamentous aggregates is seen outside the droplets after which time the solidified droplets are not in exchange with the solution. These results suggest that TDP-43CTD aggregation in phase-separating conditions is heterogeneous, with aggregation occurring first within droplets, followed by the formation of amyloids in solution from the remaining pool of monomers. Furthermore, the secondary structure content of aggregated structures inside droplets are distinct from de novo fibrils, implicating fibril polymorphism as a result of the different environments (LLPS vs. bulk solution), which may have pathological significance. In the second project, we addressed the experimental challenge of specific detection of amyloid fibrils against the background of other cellular components. Here, we demonstrate the ability to unambiguously identify cellularly internalized alpha-synuclein 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 of FCC provides a spectrally unique molecular vibration without interference from native biomolecules. Cellular uptake of FCC-alpha-synuclein 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.