Hofmeister effects on protein stability are dependent on the nature of the unfolded state

Abstract

The interpretation of salt´s effects on protein stability discriminates between low concentration regimes, dominated by ion specific-binding or Debye-Hückel screening, and high concentration regimes, generally described by Hofmeister effects. However, predicting a critical concentration at which one effect overcomes the other is still challenging. Reasonable quantitative estimates can be obtained resorting to surface/bulk solvent partition models developed for non- Coulombic effects, though these are limited by the availability of reliable structures representative of the unfolded state. Here, we use myoglobin as a model to explore how ion-dependency on the nature of the unfolded state affects protein stability, combining spectroscopic techniques with molecular dynamic simulations. To this end, the thermal and chemical stability of myoglobin was assessed, in the presence of three different salts (NaCl, (NH4)2SO4 and Na2SO4), at physiologically relevant concentrations (0-0.3 M). We observed a mild destabilization of the native state induced by each ion, attributed to unfavorable screening and hydrogen-bonds with the protein side-chains. Both effects, combined with binding of Na+, Cl- and SO42- to the thermal-unfolded state, resulted in an overall destabilization of the protein. Contrastingly, ion binding was hindered in the chemically-unfolded conformation, due to occupation of the binding sites by urea molecules. Such mechanistic action led to a lower degree of destabilization, promoting surface tension effects that overall stabilized myoglobin following the Hofmeister series. Therefore, we demonstrate that the transition concentration from Coulombic to Hofmeister effects is modulated by the nature of the unfolded state. Altogether, our findings evidence the need to characterize the structure of the unfolded state when trying to dissect the molecular mechanisms underlying effects of salts on protein stability.