Re-entrant condensation results in the formation of a condensed protein regime between two critical ion concentrations. The process is driven by neutralization and inversion of the protein charge by oppositely charged ions. Re-entrant condensation of cationic proteins by the polyvalent anions, pyrophosphate and tripolyphosphate, has previously been observed, but not for citrate, which has similar charge and size compared to the polyphosphates. Therefore, besides electrostatic interactions, other specific interactions between the polyphosphate ions and proteins must contribute. Here, we show that additional attractive interactions between arginine and tripolyphosphate determine the re-entrant condensation and decondensation boundaries of the cationic, intrinsically disordered saliva protein, histatin 5. Furthermore, we show by small-angle X-ray scattering (SAXS) that polyvalent anions cause compaction of histatin 5, as would be 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 of histatin 5, which is shown to vary with the number of arginine residues. Together, the results presented here provide further insight into an organizational mechanism that can be used to tune protein interactions in solution of both naturally occurring and synthetic proteins.

Isothermal titration calorimetry (ITC) is an apt tool for a total thermodynamic description of self-assembly of atypical amphiphiles such as anionic boron cluster compounds (COSAN) in water. Global fitting of ITC enthalpograms reveals remarkable features that differentiate COSAN from classical amphiphiles: (i) strong enthalpy and weak entropy contribution to the free energy of aggregation, (ii) low degree of counterion binding, and (iii) very low aggregation number, leading to deviations from the ideal closed association model. The counterion condensation obtained from the thermodynamic model was compared with the results of 7Li DOSY NMR of Li[COSAN] micelles, which allows direct tracking of Li cations. The basic thermodynamic study of COSAN alkaline salt aggregation was complemented by NMR and ITC experiments in dilute Li/NaCl and acetonitrile aqueous solutions of COSAN. The strong affinity of acetonitrile molecules to COSAN clusters was microscopically investigated by all-atomic molecular dynamics simulations. The impact of ionic strength on COSAN self-assembling was comparable to the behavior of classical amphiphiles, whereas even a small amount of acetonitrile cosolvent has a pronounced nonclassical character of COSAN aggregation. It demonstrates that large self-assembling changes are triggered by traces of organic solvents.

Salts are inseparable in their perturbation of molecular systems by experimental and computational methods, rendering it difficult to dissect the effects exerted by the anions and cations individually. Here we...

Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pK a value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.