Selective and specific ion binding on proteins at physiologically-relevant concentrations

Insoluble proteins dissolved in unsalted water appear to have no well-folded tertiary structures. This raises a fundamental question as to whether being unstructured is due to the absence of salt ions. To address this issue, we solubilized the insoluble ephrin-B2 cytoplasmic domain in unsalted water and first confirmed using NMR spectroscopy that it is only partially folded. Using NMR HSQC titrations with 14 different salts, we further demonstrate that the addition of salt triggers no significant folding of the protein within physiologically relevant ion concentrations. We reveal however that their 8 anions bind to the ephrin-B2 protein with high affinity and specificity at biologically-relevant concentrations. Interestingly, the binding is found to be both salt- and residue-specific.

► 83-residue cytoplasmic domain of ephrin-B2 is insoluble but can be solubilized in unsalted water.
► It is only partially-structured but the last 33 residues adopt a conformation identical to that of the fragment in buffer.
► Addition of 14 salts triggers no folding but 8 anions bind to it with high affinity at biologically-relevant concentrations.
► By eliminating the intrusion of buffer ions, we find that anion binding is both salt- and residue-specific.
► At low salt concentrations, ion-protein interactions appear to be mainly driven by electrostatics.