Charge Regulation in Intrinsically Disordered Proteins

Charge regulation is a key determinant of the net charge, conformational behavior, binding interactions, and phase equilibria of charge-rich intrinsically disordered proteins (IDPs). In this study, we examine how the apparent charge inferred from amino acid sequences can be altered through two primary mechanisms of charge regulation: proton binding to and unbinding from ionizable residues, and the preferential accumulation or exclusion of solution ions. We employ potentiometry and conductometry using ion-specific electrodes to measure the net charge of IDPs as a function of pH across different salts and salt concentrations. By extending the Nozaki-Tanford formalism for proton binding isotherms we include ion condensation effects and analyze deviations between the apparent and actual charges under various conditions. Additionally, we utilize a generalized Edwards-Muthukumar model for flexible polymers to investigate the contributions of different charge regulation modes on the conformational equilibria of IDPs. Our experimental data and computations quantify the extent of charge regulation in archetypal IDPs, including polyacids, polybases, and strong polyampholytes. The results reveal that polyampholytes with segregated blocks of oppositely charged residues are least sensitive to charge regulation, while other charge-rich IDPs display varying degrees of sensitivity. These findings provide useful insights for refining computational models and developing theories that more accurately capture the contributions of proton binding, ion condensation, and sequence-dependent effects on charge regulation in IDPs.