Ion-Pairing\nPropensity in Guanidinium Salts Dictates\nTheir Protein (De)stabilization Behavior

Since the proposition of the Hofmeister series, guanidinium\n(Gdm)\nsalts hold a special mention in protein science owing to their contrasting\neffect on protein(s) depending on the counteranion(s). For example,\nwhile GdmCl is known to act as a potential protein denaturant, Gdm<sub>2</sub>SO<sub>4</sub> offers minimal effect on protein structure.\nDespite the fact that theoretical studies reckon the formation of\nion-pairing to be responsible for such behavior, experimental validation\nof this hypothesis is still in sparse. In this study, we combine electrochemical\nimpedance spectroscopy (EIS) and THz spectroscopy to underline the\neffect of GdmCl and Gdm<sub>2</sub>SO<sub>4</sub> on a model amide\nmolecule <i>N</i>-methylacetamide (NMA). Molecular dynamics\n(MD) simulation studies predict that Gdm<sub>2</sub>SO<sub>4</sub> forms heteroion pairing in water, which inhibits Gdm<sup>+</sup> ions to approach NMA molecules, while in case of GdmCl, Gdm<sup>+</sup> ions directly interact with NMA. The experimental findings\non ion hydration, specifically the detailed analysis of the ion–water\nrattling mode, which appears in the THz frequency domain, unambiguously\nendorse this hypothesis. Our study establishes the fact that the propensity\nof ion-pairing in Gdm salts dictates their (de)stabilization effect\non proteins.