NMR Dynamic View of the Stabilization of the WW4 Domain by Neutral NaCl and Kosmotropic Na 2 SO 4 and NaH 2 PO 4 .

The Hofmeister series categorizes ions based on their effects on protein stability, yet the microscopic mechanism remains a mystery. In this series, NaCl is neutral, Na 2 SO 4 and Na 2 HPO 4 are kosmotropic, while GdmCl and NaSCN are chaotropic. This study employs CD and NMR to investigate the effects of NaCl, Na 2 SO 4 , and Na 2 HPO 4 on the conformation, stability, binding, and backbone dynamics (ps-ns and µs-ms time scales) of the WW4 domain with a high stability and accessible side chains at concentrations ≤ 200 mM. The results indicated that none of the three salts altered the conformation of WW4 or showed significant binding to the four aliphatic hydrophobic side chains. NaCl had no effect on its thermal stability, while Na 2 SO 4 and Na 2 HPO 4 enhanced the stability by ~5 °C. Interestingly, NaCl only weakly interacted with the Arg27 amide proton, whereas Na 2 SO 4 bound to Arg27 and Phe31 amide protons with Kd of 32.7 and 41.6 mM, respectively. Na 2 HPO 4 , however, bound in a non-saturable manner to Trp9, His24, and Asn36 amide protons. While the three salts had negligible effects on ps-ns backbone dynamics, NaCl and Na 2 SO 4 displayed no effect while Na 2 HPO 4 significantly increased the µs-ms backbone dynamics. These findings, combined with our recent results with GdmCl and NaSCN, suggest a microscopic mechanism for the Hofmeister series. Additionally, the data revealed a lack of simple correlation between thermodynamic stability and backbone dynamics, most likely due to enthalpy-entropy compensation. Our study rationalizes the selection of chloride and phosphate as the primary anions in extracellular and intracellular spaces, as well as polyphosphate as a primitive chaperone in certain single-cell organisms.