Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins

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.

Chain Compaction and Synergistic Destabilization of Globular Proteins by Mixture of Denaturants

“…The increase in temperature might weaken the intra‐chain interactions, thus the compressibility is reduced. Similar results are shown by both the proteins in the presence of stabilizing amino acid osmolytes as well . Hence, it is suggested that the thermal motions of surface‐exposed residues are constrained by the addition of osmolytes.…”

“…Similar results are shown by both the proteins in the presence of stabilizing amino acid osmolytes as well. [20] Hence, it is suggested that the thermal motions of surface-exposed residues are constrained by the addition of osmolytes. Also, the increase in Arg concentration decreases the K s values consistently even in the presence of Gdm.…”

Stabilization of SecA ATPase by the primary cytoplasmic salt of Escherichia coli

“…Together, these data indicate that potassium glutamate (KGlu) is the primary cytoplasmic salt in E. coli . Furthermore, we learned that KGlu can stabilize soluble proteins, because Glu − interacts unfavorably with apolar side‐chains and amide groups . Given these facts, we examined the stability of SecA in KGlu solutions.…”

“…Furthermore, we learned that KGlu can stabilize soluble proteins, because Glu − interacts unfavorably with apolar side-chains and amide groups. [19][20][21] Given these facts, we examined the stability of SecA in KGlu solutions. We report here that KGlu significantly stabilizes SecA, including its dimeric state, and increases its ATPase activity.…”

Influence of iron binding in the structural stability and cellular internalization of bovine lactoferrin

“…The major advantage of using HHP over high temperature to study protein stability is the induction of less drastic structural changes ( Perrett and Zhou, 2002 ). As a chemical approach, the use of denaturing agents such as guanidine hydrochloride (GnHCl) is also relevant, since transitions between protein conformations and other parameters such as organization and aggregation can be addressed ( Syed et al., 2018 ; Anumalla and Prabhu, 2018 ; Niether et al., 2018 ; Bian and Ji, 2014 ). Therefore, the use of these structure-disrupting agents coupled with the possibility of monitoring the transition process between the native and unfolded states is useful to assess the difference in structural stability of apo and holo-bLf – i.e., the role of iron in the structure of this protein.…”