Figure 4.

Charge reversal explains the correlation between divalent ion effects on Ddx4^N0-N4 phase separation and protein net charge

(A) Addition of CaCl₂ lowers the electrophoretic mobility ($\mu$) of Ddx4^N1 toward 0. Error bars indicate the SD.

(B) Standard curve of $\mu$ created with the Ddx4^N0-N4 charge series. Error bars indicate the SD.

(C) Upon binding, the valency mismatch between a divalent Ca²⁺ ion and a negatively charged D (Asp) or E (Glu) sidechain leads to a reversal in charge from negative (−1) to positive (+1). This change in sidechain charge upon Ca²⁺ ion binding is independent of the starting net charge of the protein.

(D) Binding of Ca²⁺ ions to Ddx4^N1 lowers the overall protein net charge. 28 ± 9 individual binding sites (negatively charged amino acid sidechains) are required to explain the change in charge observed in ELS experiments.