Effects of the protein denaturant guanidinium chloride on aqueous hydrophobic contact-pair interactions.

Guanidinium chloride (GdmCl) is one of the most common protein denaturants. Although GdmCl is well known in the field of protein folding, the mechanism by which it denatures proteins is not well understood. In fact, there are few studies looking at its effects on hydrophobic interactions. In this work the effect of GdmCl on hydrophobic interactions has been studied by observing how the denaturant influences model systems of phenyl and alkyl hydrophobic contact pairs. Contact pair formation is monitored through the use of fluorescence spectroscopy, i.e., measuring the intrinsic phenol fluorescence being quenched by carboxylate ions. Hydrophobic interactions are isolated from other interactions through a previously developed methodology. The results show that GdmCl does not significantly affect hydrophobic interactions between small moieties such as methyl groups and phenol; while on the other hand, the interaction of larger hydrophobes such as hexyl and heptyl groups with phenol is significantly destabilized.

Effects of the osmolyte TMAO (Trimethylamine-N-oxide) on aqueous hydrophobic contact-pair interactions.

Osmolytes are small, soluble organic molecules produced by living organisms for maintaining cell volume. These molecules have also been shown to have significant effects on the stability of proteins. Perhaps one of the most studied osmolytes is Trimethylamine-N-oxide (TMAO). Thermodynamic studies of the effects of TMAO on proteins have shown that this molecule is a strong stabilizer of the protein folded state, thus being able to counteract the effects of protein denaturants such as urea and guanidine hydrochloride. Most studies of TMAO effects on bio-molecular stability have until now been focused on how the osmolyte reduces the solubility of polypeptide backbones, while the effects of TMAO on hydrophobic interactions are still not well understood. In fact, there are few experimental data measuring the effect of TMAO on hydrophobic interactions. This work studies phenyl and alkyl contact pairs as model hydrophobic contact pairs. The formation of these contact pairs is monitored using fluorescence, i.e., through the quenching of phenol fluorescence by carboxylate ions; and a methodology is developed to isolate hydrophobic contributions from other interactions. The data demonstrate that the addition of TMAO to the aqueous solvent destabilizes hydrophobic contact pairs formed between alkyl and phenyl moieties. In other words, TMAO acts as a "denaturant" for hydrophobic interactions.