Zwitterionic osmolytes are widely known to have a protein-protective effect against high salt concentration, but a mechanistic picture of osmolyte function remains elusive. Here total scattering is used to determine the room temperature liquid structure of two model cytosol solutions containing trimethylglycine (TMG) with either sodium or potassium chloride. H/D isotopic substitution is used to obtain differential neutron scattering cross sections at multiple contrasts in addition to an X-ray structure factor, and an Empirical Potential Structure Refinement (EPSR) simulation is fitted to the experimental data. We reveal the nature of the interaction between TMG molecules and ions in solution, observing binding between cations and the TMG carboxyl group. We observe three key specific ion effects: first, that sodium ions are more tightly localised at the carboxyl group; second, that sodium localisation in turn promotes head-to-head bridging between carboxyl groups when compared to potassium or no added ions, resulting in strong oxygen-oxygen correlations; and third, that sodium ions promote TMG clusters with greater orientational order, more fully shielding the ion but also in turn limiting access to the carboxyl groups for other molecules. These observations have implications for the bioavailability and protein-stabilising effect of osmolytes under changing extracellular salt conditions.
