No abstract

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two-state transitions during chemical denaturation. The extent of stability exerted by Glu is higher for RNase A at higher temperature, whereas it provides more stability for α-LA at lower temperature. Thus, the experiments indicate that Glu induces a thermal equilibrium intermediate and increases the thermodynamic stability of proteins irrespective of their surface charges. The extent of stability varies between the proteins in a temperature-dependent manner.

No abstract

Characterization of unfolded states is essential to understand the mechanism of protein folding. This study examines the unfolded states of RNase A and α‐LA in the mixture of ′′structurally‐similar′′ chemical denaturants, arginine (Arg) and guanidinium chloride (Gdm). Thermal‐ and chemical‐unfolding experiments show that the mixture of denaturants synergistically destabilizes the proteins. Volumetric analysis shows that partial molar volume (V°) and adiabatic compressibility (Ks) of the proteins decreases with increasing [Gdm]. This suggests that the proteins might be gradually losing their intra‐molecular interactions resulting in decreased internal cavity. The addition of Arg to the Gdm‐unfolded states decreases both V° and Ks values indicating compaction of the protein chains. This could be attributed to reduction in the hydration of surface‐exposed residues of the proteins upon direct interaction with Arg and increased non‐native contacts. The results provide a direct experimental evidence for the compaction of unfolded protein chain upon addition of another denaturant.