Synucleinopathies such as Parkinson’s disease (PD) are characterized by the development of Lewy bodies—intracellular protein aggregates in neurons. The principal component of these inclusions is α-synuclein (α-syn), a protein that is natively unstructured but forms insoluble amyloid fibrils in the disease state. Although methods for study of amyloid formation in vitro are robust, the structural plasticity of α-syn and its sensitivity to perturbation present a significant challenge in the complex cellular milieu. Hence, new approaches are needed to illuminate the behavior of α-syn in vivo. Recent work in our laboratory has developed Raman spectral imaging to study α-syn in cultured cells, combining the spatial resolution of confocal microscopy with the rich structural information of vibrational spectroscopy. In this study, we leverage an evolved aminoacyl tRNA synthetase/tRNA pair (“21st pair technology”) to site-specifically incorporate the unnatural amino acid 4-ethynyl-L-phenylalanine (FCC) as a Raman vibrational probe into α-syn. The alkyne stretching band appears in a spectral region devoid of background interference by other biomolecules and is sensitive to solvation changes, enabling the unambiguous localization of α-syn while simultaneously probing the local environment of the sidechain. This approach will permit direct comparison of in vitro and in cellulo data as the same construct can be expressed in both bacterial and mammalian cells, allowing the introduction of FCC at any position of choice and evaluation of conformational changes of α-syn in a cellular environment.

Genetically encoding a terminal alkyne as a site-specific Raman probe of α-synuclein aggregation

ACS Spring Meeting 2021

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