α-Synuclein (αS) is a 140 amino acid intrinsically disordered protein (IDP) that is abundant in neurons and is the primary component of Lewy bodies, the aberrant intracellular aggregates that are present in the brains of Parkinson’s disease (PD) patients. In the synapse, αS has proposed functions in SNARE-complex assembly, neurotransmitter trafficking, and synaptic vesicle binding, although molecular details of these are poorly understood. αS is subject to a variety of post-translational modifications (PTMs). With the exception of N-terminal acetylation, αS acetyl , which is constitutive, PTMs are reversible side chain modifications which are likely to impact αS structure and function. Among the regulatory PTMs, two of particular interest are phosphorylation at S129 (pS129) or Y39 (pY39). While pS129 is a prevalent biomarker for PD, it also alters the cellular localization of soluble αS in neurons. In contrast, pY39 is located within the central helical region of membrane-bound αS and may alter the membrane-bound function. Using a combination of bacterial co-expression, enzymatic phosphorylation, unnatural amino acid incorporation, and native chemical ligation, I have produced αS acetyl that is also modified by pS129, pY39, or both. I used a combination of single molecule Forster resonance energy transfer and fluorescence correlation spectroscopy to determine how these modifications impact the conformational ensemble of αS in solution and upon interactions with binding partners.
Castrodad et al. (Sun,) studied this question.