Hofmeister Effects of Group II Cations as Seen in the Unfolding of Ribonuclease A

Graphical Abstract

Group II cations affect protein folding through two important modalities: (top) promote protein folding by enhancing the hydrophobic effect through reduction of solvent void volume; (bottom) promote protein unfolding by forming contact pairs with amide carbonyls that help solubilize the protein amide backbone.

Abstract

This work studies the effects of alkaline-earth cation addition on the unfolding free energy of a model protein, pancreatic Ribonuclease A (RNase A) by differential scanning calorimetry analysis. RNase A was chosen because: a) it does not specifically bind Mg²⁺, Ca²⁺ and Sr²⁺ cations and b) maintains its structural integrity throughout a large pH range. We have measured and compared the effects of NaCl, MgCl₂, CaCl₂ and SrCl₂ addition on the melting point of RNase A. Our results show that even though the addition of group II cations to aqueous solvent reduces the solubility of nonpolar residues (and enhances the hydrophobic effect), their interactions with the amide moieties are strong enough to “salt-them-in” the solvent, thereby causing an overall protein stability reduction. We demonstrate that the amide-cation interactions are a major contributor to the observed “Hofmeister Effects” of group II cations in protein folding. Our analysis suggests that protein folding “Hofmeister Effects” of group II cations, are mostly the aggregate sum of how cation addition simultaneously salts-out hydrophobic moieties by increasing the cavitation free energy, while promoting the salting-in of amide moieties through contact pair formation.