Comparison of strategies for non-perturbing labeling of α-synuclein to study amyloidogenesis

Characterization of the amyloidogenic Parkinson's disease protein alpha-synuclein (alpha S) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine alpha S fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled alpha S were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled alpha S can then be used to monitor conformational changes during fibril formation or cellular uptake of alpha S fibrils in models of disease propagation.