Does Urea Preferentially Interact with Amide Moieties or Nonpolar Sidechains? A Question Answered Through a Judicious Selection of Model Systems

Abstract

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.

Computational Enzyme Redesign Enhances Tolerance to Denaturants for Peptide C-Terminal Amidation

“…However, accurately assessing the interactions between surface hydrophobic residues, especially aromatic ones, and solvent molecules remains a challenging task in computation protein redesign. It’s imperative to acknowledge that urea and Gnd + ions can interact not only with hydrophilic but also hydrophobic side chains . Recent computational modeling studies have advanced the notion that Gnd + ions may engage in water-assisted cation–π interactions with individual aromatic amino acids .…”

“…It’s imperative to acknowledge that urea and Gnd + ions can interact not only with hydrophilic but also hydrophobic side chains. 47 Recent computational modeling studies have advanced the notion that Gnd + ions may engage in water-assisted cation–π interactions with individual aromatic amino acids. 48 Nonetheless, due to the intrinsic chemical and topographical heterogeneity of the protein surface, these interactions are notably influenced by the hydration patterns of Gnd + ions and the relative orientations between Gnd + ions and the aromatic rings.…”

The Streptococcus phage protein paratox is an intrinsically disordered protein

“…This scenario can also explain the consistency observed between the values obtained through urea denaturation and the salt and osmolyte titration experiments. Urea unfolds proteins mostly through dispersion interactions rather than electrostatics (Ajayi et al, 2023 ), therefore would be minimally affected by urea addition.…”

“…This scenario can also explain the consistency observed between the $$$ \Delta {G}_{\left[\mathrm{salt}\right]=0}^{\mathrm{unfolding}} $$$ values obtained through urea denaturation and the salt and osmolyte titration experiments. Urea unfolds proteins mostly through dispersion interactions rather than electrostatics (Ajayi et al, 2023), therefore $$$ \Delta \Delta {G}_{\left[\mathrm{salt}\right]}^{\mathrm{electrostatic}} $$$ would be minimally affected by urea addition.…”