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Post-translational modifications (PTMs) impact the pathological aggregation of α-synuclein (αS), a hallmark of Parkinson's disease (PD). Here, we synthesize αS phosphorylated at tyrosine 39 (pY 39 ) through a novel route using in vitro enzymatic phosphorylation of a fragment followed by ligation to form the full-length protein. We can execute this synthesis in combination with unnatural amino acid mutagenesis to include two fluorescent labels for Förster resonance energy transfer (FRET) studies. We determine the effect of pY 39 on the aggregation of αS and compare our authentically phosphorylated material to the corresponding glutamate 39 "phosphomimetic." Intriguingly, we find that αS-pY 39 can either accelerate or decelerate aggregation, depending on the fraction of phosphorylated protein. The αS-E 39 mutant can qualitatively reproduce some, but not all, of these effects. FRET measurements and analysis of existing structures of αS help to provide an explanation for this phenomenon. Our results have important implications for the treatment of PD patients with tyrosine kinase inhibitors and highlight the importance of validating phosphomimetics through studies of authentic PTMs.

Growing evidence implicates α-synuclein aggregation as a key driver of neurodegeneration in Parkinson's disease (PD) and other neurodegenerative disorders. Herein, the molecular and structural mechanisms of inhibiting α-synuclein aggregation by novel analogs of nordihydroguaiaretic acid (NDGA), a phenolic dibenzenediol lignan, were explored using an array of biochemical and biophysical methodologies. NDGA analogs induced modest, progressive compaction of monomeric α-synuclein, preventing aggregation into amyloid-like fibrils. This conformational remodeling preserved the dynamic adoption of α-helical conformations, which are essential for physiological membrane interactions. Oxidation-dependent NDGA cyclization was required for the interaction with monomeric α-synuclein. NDGA analog-pretreated α-synuclein did not aggregate even without NDGA-analogs in the aggregation mixture. Strikingly, NDGA-pretreated α-synuclein suppressed aggregation of naïve untreated aggregation-competent monomeric α-synuclein. Further, cyclized NDGA reduced α-synuclein-driven neurodegeneration in Caenorhabditis elegans. The cyclized NDGA analogs may serve as a platform for the development of small molecules that stabilize aggregation-resistant α-synuclein monomers without interfering with functional conformations yielding potential therapies for PD and related disorders.

Accurate imaging of nanometer-sized structures and morphologies is essential to characterizing amyloid species formed at various stages of amyloid aggregation. In this article, we examine the effect of different drying procedures on the final morphology of surface-mediated fibrils formed during the incubation period, which may then be mistaken as oligomers or protofibrils intentionally formed in solution for a particular study. Atomic force microscopy results show that some artifacts, such as globules, flakelike structures, and even micrometer-long fibrils, can be produced under various drying conditions. We also demonstrate that one can prevent drying artifacts by using an appropriate spin-coating procedure to dry amyloid samples. This procedure can bypass the wetting/dewetting transition of the liquid layer during the drying process and preserve the structure of interest on the substrate without generating drying artifacts.

Characterization of the amyloidogenic Parkinson's disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS 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 αS 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 αS can then be used to monitor conformational changes during fibril formation or cellular uptake of αS fibrils in models of disease propagation.