Not All Charges Are Equal: Side-Chain Chemistry Reshapes the Disordered Ensemble of α-synuclein

The conformational ensembles of intrinsically disordered proteins (IDPs) are governed by the balance of electrostatic and hydrophobic interactions encoded in their primary sequences. Current polymer-physics models of IDPs frequently group amino acids by coarse-grained properties, such as net charge, often overlooking the distinct residues side-chain chemistries. Analysis across the IDP database reveals that these sequences are sensitive to specific residue identities, where the substitution of aromatic, proline, or hydrophobic groups serves as a primary driver of chain dimensions. However, these data also highlight that even subtle chemical variations between similarly charged residues can consistently shift global compaction. Here, we explore the role of residue identity using small angle X-ray scattering (SAXS) of seven -synuclein variants with progressively increasing numbers of lysine-to-arginine substitutions, two positively charged amino acids with different side-chain chemistry. We show that increasing arginine content drives a systematic compaction of the conformational ensemble, although variants with identical number of substitutions but different positional arrangements suggest influence to the sequence context. Moreover, while increasing salt concentrations shift the structural ensemble from a Gaussian toward a self-avoiding-walk statistics, the arginine-dependent contraction trend remains robust across both regimes. Molecular-dynamics simulations combined with SAXS data reveal that arginine substitutions reduce ensemble heterogeneity by stabilizing transient long-range contacts. Finally, aggregation assays demonstrate that this arginine-driven compaction correlates with an accelerated transition to amyloid fibrils. Our findings demonstrate that chemically subtle substitutions between similarly charged residues can fundamentally reshape the conformational ensemble of IDPs, suggesting that side-chain identity is a critical, yet underappreciated, determinant of protein disorder and proteotoxicity. O_TEXTBOXSignificance StatementIntrinsically disordered proteins (IDPs) are critical to cellular signaling and neurodegeneration, yet our ability to predict their behavior remains limited by a "coarse-grained" understanding of their sequences. We expand the view that net charge and patterning is a primary determinant of IDP dimensions by showing that lysine and arginine, residues identical in charge, exert opposite effects on the conformational landscape of -synuclein model-system. We find that arginine substitutions act as a "molecular glue", driving protein compaction and reducing ensemble heterogeneity. Crucially, this compaction accelerates amyloid aggregation, overturning the conventional assumption that collapsed states protect against fibrillization. This work demonstrates that side-chain identity and patterning are vital for protein homeostasis, providing a new framework for the rational design of IDP-based therapeutics. C_TEXTBOX