In aqueous solutions proteins can undergo a variety of different phase behaviours, inlcuding liquid-liquid phase separation and reentrant condensation. The latter process has been observed in solutions containing charged globular proteins in the presence of oppositely charged multivalent ions. Reentrant condensation is characterised by the formation of a condensed protein regime between two critical ion concentrations. Formation of the condensed regime is thought to be driven by neutralization and inversion of the protein charge by the ion. Similarly, decondensation at the upper boundary is caused by overcharging of the protein by the ion, resulting in long-range electrostatic repulsion between protein molecules. Recently, reentrant condensation of the cationic globular protein lysozyme by the polyvalent anions pyrophosphate and tripolyphosphate, but not by cirtrate was observed. Despite the fact that citrate has similar charge and size to pyrophosphate. Therefore, besides electrostatic interactions, other specific interactions between the polyphosphate ions and the protein, contribute. Here, we show that additional attractive interactions between arginine residues and tripolyphosphate determine the reentrant condensation and decondensation boundaries of the cationic, intrinsically disordered saliva protein, Histatin 5. Furthermore, we show by SAXS that polyvalent anions cause compaction of Histatin 5, above what is expected based solely on electrostatic interactions. Hence, we conclude that arginine-phosphate specific interactions not only regulate solution properties but also influence the conformational ensemble, which, is shown to vary with the number of arginine residues. Together the results presented here provide further insight into a organizational mechanism that can be used to tune protein interactions in solution of both naturally occurring and synthetic proteins.

The impact of arginine-phosphate interactions on the reentrant condensation of the intrinsically disordered histatin 5 protein

ACS Spring Meeting 2021

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