Biology subjects

Abakah, B.

Publications and source records attributed to Abakah, B..

2 recordsLinked to original sources

bioRxiv2026

Genetic Code Expansion, Enzymatic Modification, and C-Terminal Labeling Enable Facile Production of Highly Modified α-Synuclein

-Synuclein (S), a protein that plays a central role in Parkinsons disease and related synucleinopathies, is an intrinsically disordered protein (IDP) whose functional interactions and aggregation behavior can be strongly influenced by post-translational modifications (PTMs). Phosphorylation, acetylation, and other PTMs regulate Ss interactions with lipid membranes and binding partners, whereas their dysregulation is associated with aggregation and neuronal toxicity. Despite significant progress through chemical and semi-synthetic approaches, investigating the combinatorial effects of PTMs has remained challenging due to the lack of accessible, site-specific methods. Here, we present an integrated strategy combining genetic code expansion, enzymatic modification, and intein-mediated click chemistry to generate S variants bearing multiple defined PTMs and a C-terminal fluorescent label. The resulting constructs enable direct evaluation of how individual and combined PTMs influence S structure, lipid binding, and cellular internalization. Our approach expands the molecular toolkit for dissecting PTM crosstalk in S and other aggregation-prone IDPs, advancing mechanistic understanding and supporting the development of therapeutic strategies for neurodegenerative disease.

biochemistry↗

bioRxiv2025

Genetic Code Expansion and Enzymatic Modifications as Accessible Methods for Studying Site-Specific Post-Translational Modifications of Alpha-Synuclein and Tau

Alpha-synuclein (S) and tau play important roles in the pathology of Parkinsons Disease and Alzheimers Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of S and tau monomers, as well as their propensity to aggregate. While there have been many valuable insights into site-specific effects of PTMs garnered through chemical synthesis and semi-synthesis, these techniques are often outside of the expertise of biochemistry and biophysics laboratories wishing to study S and tau. Therefore, we have assembled a primer on genetic code expansion and enzymatic modification approaches to installing PTMs into S and tau site-specifically, including isotopic labeling for NMR and fluorescent labeling for biophysics and microscopy experiments. These methods should be enabling for those wishing to study authentic PTMs in S or tau as well as the broader field of IDPs and aggregating proteins.

biochemistry↗