Tau is an intrinsically disordered protein implicated in the neurodegenerative disorders classified as tauopathies, such as Alzheimer’s disease, Pick’s disease, and chronic traumatic encephalopathy (CTE). Tau plays a role in maintaining microtubule dynamics and binds both to soluble tubulin dimers and to microtubules. Post-translational modifications (PTMs) regulate these interactions, and dysregulation of PTMs may predispose Tau to oligomerization and aggregation. We utilized native chemical ligation (NCL), a method of ligating peptides obtained by a combination of solid phase synthesis and recombinant expression to produce full-length protein bearing phosphate groups at specific sites. This approach allows us to compare the effects of phosphomimic mutations and authentically phosphorylated constructs in microtubule polymerization assays. By combining the NCL approach with amber codon expression, we introduced the unnatural amino acid propargyltyrosine (PpY) to fluorescently label Tau. Fluorescently labeled Tau was used to quantify binding to soluble tubulin via fluorescent correlation spectroscopy (FCS). By employing these techniques, we aim to gain insights into the molecular mechanisms underlying Tau function and its interactions with tubulin, which are crucial for understanding loss of function that occurs in neurodegenerative diseases.
Tomlinson et al. (Sun,) studied this question.