Shedding light on the extra thermal stability of thermophilic proteins

Abstract

An entropic stabilization mechanism has recently gained attention and credibility as the physical ground for the extra thermal stability of globular proteins from thermophilic microorganisms. An empirical result, obtained from the analysis of thermodynamic data for a large set of proteins, strengthens the general reliability of the theoretical approach originally devised to rationalize the occurrence of cold denaturation [Graziano, PCCP 2014, 16, 21755-21767]. It is shown that this theoretical approach can readily account for the entropic stabilization mechanism. On decreasing the conformational entropy gain associated with denaturation, the thermal stability of a model globular protein increases markedly.

Effect of heavy water on the conformational stability of globular proteins

“…The devised theoretical approach leads to the following expression for Δ G d : where Δ G c (D‐state) and Δ G c (N‐state) represent the reversible work to create in H 2 O or D 2 O a cavity suitable to host the D‐state and the N‐state, respectively; Δ S conf represents the conformational entropy gain of the polypeptide chain upon denaturation; E a (D‐state) and E a (N‐state) represent the energetic interactions (i.e., both van der Waals attractions and H‐bonds) among the D‐state or the N‐state and surrounding H 2 O or D 2 O molecules; Δ E (intra) is the difference in intrachain energetic interactions between the D‐state and the N‐state. In the Gibbs energy balance of Equation , terms due to the reorganization of H‐bonds among H 2 O or D 2 O molecules are not present because the latter process is characterized by an almost complete enthalpy‐entropy compensation .…”

“…In the Gibbs energy balance of Equation , terms due to the reorganization of H‐bonds among H 2 O or D 2 O molecules are not present because the latter process is characterized by an almost complete enthalpy‐entropy compensation . The ΔΔ G c term is always positive and stabilizes the N‐state, because it is more costly to create a cavity suitable to host the D‐state, which has a water accessible surface area, WASA, larger than that of the N‐state (it has to be underscored that WASA is the physically correct measure of the solvent‐excluded volume effect caused by a cavity or a molecule); the – T ⋅Δ S conf term is always negative and stabilizes the D‐state; the Δ E a term has been assumed to be equal to zero in H 2 O for a globular protein that exactly corresponds to the assumptions of the theoretical approach (i.e., a “model” globular protein) …”

“…The ΔΔ G c term is calculated by means of the analytical relationships provided by classic scaled particle theory, SPT, assuming that: (a) the N‐state is modeled by a sphere and the D‐state is modeled by a prolate spherocylinder whose van der Waals volume is equal to that of the sphere modeling the N‐state; (b) the geometric features of the two states are not affected on passing from H 2 O to D 2 O. Specifically, reliable numbers for a 138‐residue globular protein are the following: the N‐state is a sphere of radius a = 15 Å, V vdW = 14137 Å 3 and WASA = 3380 Å 2 (calculated using for water molecules the customary radius of 1.4 Å), whereas the D‐state is a prolate spherocylinder of radius a = 6 Å, cylindrical length l = 117 Å, V vdW = 14137 Å 3 and WASA = 6128 Å 2 . The fact that about 50% of WASA is buried upon folding of the polypeptide chain is in line with available structural information, considering that the D‐state resembles a compact prolate ellipsoid .…”

“…The T ⋅Δ S conf term is calculated in the assumption that each residue gains an average, temperature‐independent, conformational entropy upon denaturation: where N res = 138 and Δ S conf (res) = 19.0 J K −1 molres −1 . The latter number is considered to be a reliable value for a “model” globular protein because it is close to the estimate obtained by Sosnick and coworkers, Δ S conf (res) = 19.5 J K −1 molres −1 , exploiting realistic statistical ensembles for both the N‐state and the D‐state of ubiquitin at 300 K and 1 atm, constructed by means of MD simulations.…”

“…We have devised a statistical thermodynamic approach that has been proven able to rationalize several features of the conformational stability of globular proteins in water: (a) the geometric nature of the hydrophobic effect, originating from the decrease in solvent‐excluded volume associated with chain folding, that leads to an increase in translational entropy of water molecules (i.e., it is the driving force of folding); (b) the parabola‐like shape of the trend of Δ G d versus temperature, called the stability curve of a globular protein, indicating the occurrence of both cold and hot denaturations, and providing a molecular level insight into their origin; (c) the physical meaning of the temperature, labeled T H , at which the denaturation enthalpy change, Δ H d , is zero and its relationship with the temperature of maximum density of water; (d) the entropic nature of the extra‐stability of thermophilic proteins . In this study, we would like to apply the same theoretical approach to clarify the stabilizing effect of heavy water.…”