Role of Urea–Aromatic Stacking Interactions in Stabilizing the Aromatic Residues of the Protein in Urea-Induced Denatured State

Abstract

A delicate balance of different types of intramolecular interactions makes the folded states of proteins marginally more stable than the unfolded states. Experiments use thermal, chemical, or mechanical stress to perturb the folding equilibrium for examining protein stability and the protein folding process. Elucidation of the mechanism by which chemical denaturants unfold proteins is crucial; this study explores the nature of urea−aromatic interactions relevant in urea-assisted protein denaturation. Free energy profiles corresponding to the unfolding of Trp-cage miniprotein in the presence and absence of urea at three different temperatures demonstrate the distortion of the hydrophobic core to be a crucial step. Exposure of the Trp6 residue to the solvent is found to be favored in the presence of urea. Previous experiments showed that urea has a high affinity for aromatic groups of proteins. We show here that this is due to the remarkable ability of urea to form stacking and NH−π interactions with aromatic groups of proteins. Urea−nucleobase stacking interactions have been shown to be crucial in urea-assisted RNA unfolding. Examination of these interactions using microsecond-long unrestrained simulations shows that urea−aromatic stacking interactions are stabilizing and long lasting. Further MD simulations, thermodynamic integration, and quantum mechanical calculations on aromatic model systems reveal that such interactions are possible for all the aromatic amino acid side-chains. Finally, we validate the ubiquitous nature of urea−aromatic stacking interactions by analyzing experimental structures of urea transporters and proteins crystallized in the presence of urea or urea derivatives.

Energetic, Structural and Dynamic Properties of Nucleobase-Urea Interactions that Aid in Urea Assisted RNA Unfolding

“…This suggests that the stabilizing nature of nucleobases in their extrahelical conformation in aqueous urea is largely due to dispersive type interactions between urea and nucleobase. Notably, the primary component of the difference of the interaction energies of unfolded and folded states of protein with urea was reported to be dispersion type interactions 18,37–40 . Significantly lower dispersive type interactions in pure water explain the denaturation of RNA in the presence urea.…”

Synthesis of Urea Derivatives from CO2 and Silylamines

“…Urea derivatives have found a myriad of applications in biological studies, analytical chemistry, pharmaceuticals, polymer sciences, and agrochemicals . Recent examples include their use in the preparation of lactone derivatives that serve as bacteria inhibitors, while sulfonylureas are used as drugs to treat type II diabetes . Alternatively, anionic ureas have been exploited in ring‐opening polymerizations for materials and organocatalysis applications, while bulky ureas have been used as precursors to guanidinate ligands .…”

Stacking Interactions of Heterocyclic Drug Fragments with Protein Amide Backbones

“…Among these are amide stacking interactions, which are the attractive interactions between arenes and the π‐system of an amide. Amide stacking interactions are stronger than many other stacking interactions, and can be competitive with hydrogen bonds; they can also play a role in protein structure and stability, and similar interactions have been implicated in urea‐induced denaturation of proteins . Unlike stacking interactions of arenes, amide stacking is still relatively poorly understood.…”