Leveraging structurally-informed bioorthogonal conjugation for alpha synuclein fibril surveillance

Abstract

Alpha synuclein (-syn) amyloid fibrils are etiological agents of neurodegenerative disease. To better understand the molecular and cellular basis for their persistence and spread, we implemented a fluorophore labeling strategy to track pre-formed recombinant -syn fibrils in solution and in cells. We leveraged amber codon mediated incorporation of a tetrazine-based artificial amino acid (TetV2.0) to install a strained trans-cyclooctene (sTCO)-conjugated Janeliafluor (JF549), at four structurally-informed sites on human -syn: residues 4, 60, 96 and 136. A structure of fibrils labeled at residue 60 showed density consistent with TetV2.0 incorporation and a core structure similar to recombinant type 1M -syn fibrils. Coupling of TetV2-containing fibrils to sTCO-JF549 proceeded under mild buffer conditions and in the presence of the disease-associated cofactor and cytotoxic lipid, psychosine. Labeled psychosine-induced fibrils retained their polymorph-specific features during labeling; their structure resembled that of type 1A recombinant -syn fibrils and showed a solvent exposed site at residue 60, amenable to bioorthogonal conjugation. Labeled fibrils also seeded new fibril growth in vitro, and induced the formation of puncta in -syn FRET biosensor HEK293T cells, facilitating the simultaneous tracking of exogenous and endogenous -syn aggregates and their localization within the cells. This approach opens a correlative bridge between fibril structure and function in living systems.