Destabilization of Hydrophobic Core of Chicken Villin Headpiece in Guanidinium Chloride Induced Denaturation: Hint of π-Cation Interaction

Abstract

Despite their routine use as protein denaturants, the comprehensive understanding of the molecular mechanisms by which urea and guanidinium chloride (GdmCl) disrupts proteins' structure is still lacking. Here, we use steered molecular dynamics simulations along with the umbrella sampling technique to elucidate the mechanism of unfolding of chicken villin headpiece (HP-36) in these two denaturants. We find that while urea denatures protein predominantly by forming hydrogen bonds with the protein backbone, GdmCl commences unfolding by weakening of the hydrophobic interactions present in the core. The potential of mean force calculation indicates the reduction of hydrophobic interactions between two benzene moieties in 6 M GdmCl as compared to 6 M urea. We observe a near parallel orientation between the guanidinium cation and aromatic side chains of the HP-36 suggesting π-cation type stacking interactions which play a crucial role in weakening of the hydrophobic interaction. We use QM/MM optimization calculations to estimate the energetics of this π-cation interaction. Additionally, the consistency of the unfolding paths between high temperature (400 K) unfolding simulations and steered molecular dynamics simulations strengthens the proposed molecular mechanism of unfolding further.

Exploring the role of hydrophilic amino acids in unfolding of protein in aqueous ethanol solution

“…The compact core of a protein can be disrupted by several external factors such as change in temperature, pressure 26‐29 etc. It can also be altered by adding external denaturants like urea, GdmCl 30‐32 etc. Several interesting studies have been performed till date regarding the effect of co‐solvents on the structure of proteins and hydrophobes 33‐45 .…”

“…52 On the other hand, aqueous urea solution as well as aqueous GdmCl solution is known to denature protein structure appreciably but the mechanism by which they do so vastly differs from each other. 30 While urea is known to denature protein via forming hydrogen bonds with protein backbone, GdmCl initiates unfolding by weakening the core hydrophobic interaction of the protein. In the similar context, the behavior of a protein in the presence of protecting osmolytes like TMAO, trehalose etc also draws marked attention.…”

“…Often it is found that small molecule osmolytes exhibit diverse effect on protein structure and stability. Certain aqueous mixtures are found to destabilize protein structure significantly [30][31][32] whereas in some cases the reverse effect is noteworthy. [46][47][48][49] Also, the stabilization and destabilization of the structure of a protein in aqueous binary mixture is found to be composition dependent in many cases.…”

Protein unfolding mechanisms and their effects on folding experiments

“…The molecular dynamics of an unfolded protein indicate that urea readily forms hydrogen bonds with the peptide backbone, disrupts native contacts, and makes extended conformations favorable 11 . Simulations comparing urea and guanidine on the same protein find that guanidine does not make many hydrogen bonds but does disrupt hydrophobic interactions within the native state, particularly between aromatic side chains 12 .…”

Towards Rational Chemosensor Design through Improved Understanding of Experimental Parameter Variation and Tolerance in Cyclodextrin-Promoted Fluorescence Detection

“…Salts are known to influence inclusion complexation by a variety of pathways, including through the disruption of intermolecular hydrogen bonding, the formation of association complexes with hydrophobic and/or charged analytes, and the facilitation of ternary complex formation [55][56][57]. Their presence also affects hydrophobic association, with salts such as sodium chloride increasing hydrophobic binding [58], and salts such as guanidinium hydrochloride (GuHCl) decreasing the hydrophobic interactions [59]. Fluorescence energy transfer in these cyclodextrin systems was measured at various salt concentrations (1 M guanidium hydrochloride and 1 M sodium chloride for all analytes and 0.1 M and 2 M concentrations for select analytes) to determine the robustness of the system to operate under conditions known to disrupt important intermolecular interactions.…”