The transfer model suggests that urea unfolds proteins mainly by increasing the solubility of the amide backbone, probably through urea-induced increase in hydrogen bonding. Other studies suggest that urea addition increases the magnitude of solvent-solute van der Waals interactions, which increases the solubility of nonpolar sidechains. More recent analyses hypothesize that urea has a similar effect in increasing the solubility of backbone and sidechain groups. In this work, we compare the effects of urea addition on the solvation of amides and alkyl groups. At first, we study the effects of urea addition upon solvent hydrogen bonding acidity and basicity through the perturbation in the fluorescence spectrum of probes 1-AN and 1-DMAN. Our results demonstrate that the solvent's hydrogen bonding properties are minimally affected by urea addition. Subsequently, we show that urea addition does not perturb the intra-molecular hydrogen bonding in salicylic acid significantly. Finally, we investigate how urea preferentially interacts with amide and alkyl groups moieties in water by comparing the effects of urea addition upon the solubility of acetaminophen and 4-tertbutylphenol. We show that urea affects amide and tbutyl solubility (lowers the transfer free energy of both amide (backbone) and alkyl (sidechain) groups) in a similar fashion. In other words, preferential interaction of urea with both moieties contributes to protein denaturation.

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 Mg 2 + , Ca 2 + and Sr 2 + cations and b) maintains its structural integrity throughout a large pH range. We have measured and compared the effects of NaCl, MgCl 2 , CaCl 2 and SrCl 2 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.