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

Abstract

Edited by Miguel De la Rosa Keywords:Insoluble protein Ephrin-B2 Intrinsically unstructured protein Protein-ion interaction Hofmeister series NMR spectroscopy a b s t r a c t 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.

Why do proteins aggregate? “Intrinsically insoluble proteins” and “dark mediators” revealed by studies on “insoluble proteins” solubilized in pure water

“…We thus extended our previous investigations by titrating the 83-residue cytoplasmic domain of ephrin-B2 with 14 salts whose 8 anions are located in the middle, on the left and right sides of the Hofmeister series 96 . Previously the entire domain was found to be insoluble and consequently its structure remains unstudied 97 .…”

“…To determine the relative contribution of cations and anions to the HSQC peak shifts induced by salts, we monitored the shifts by titrating chloride salts with different cations, including MgCl 2 , KCl, CaCl 2 , and GdmCl. The different chloride salts caused very similar patterns of HSQC peak shifts, suggesting that the observed effects are mostly due to the chloride anion 96 . These results suggest that the HSQC peak shifts observed here are mostly triggered by the asymmetric binding of different anions to the protein residues.…”

“…Our study on the unstructured ephrin-B2 cytoplasmic domain 96 suggests that the protein-anion interaction is mostly modulated by anion type, protein conformation and electrostatic property. Therefore, a high-resolution study of the effects of different salts on a well-folded protein is crucial.…”

“…To our surprise again, the majority of the apparent Kd values are found to be less than 50 mM, thus indicating that all eight anions bind to the ephrin-B2 cytoplasmic domain with high affinity. Of particular significance, Na 2 SO 4 is found to be the strongest binder to most residues, with an average apparent Kd of only 1 mM 96 .…”

Charge matters

“…It has been shown that reasonable estimates of z P are obtained from electrophoretic measurements for RNase A (Moody et al 2005) and hen egg white lysozyme (Gokarn et al 2011), both of which exhibit monovalent anion binding. Song's group at the University of Singapore have characterized monovalent anion binding by NMR (Miao et al 2011). Both z eff and NMR measurements reveal that the relative binding strength of anions follows the Hofmeister series (Gokarn et al 2011), suggesting that charge neutralization may be an important contributor to solubility.…”