Solvent denaturation and stabilization of globular proteins

Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the effects of solvents on protein stability. Folding is assumed to be driven by solvophobic interactions and opposed by conformational entropy. The solvent dependence of the solvophobic interactions is taken from transfer experiments of Nozaki and Tanford on amino acids into aqueous solutions of urea or guanidine hydrochloride (GuHCl). On the basis of the assumption of two pathways involving collapse and formation of a core, the theory predicts that increasing denaturant should lead to a two-state denaturation transition (i.e., there is a stable state along each path separated by a free energy barrier). The denaturation midpoint is predicted to occur at higher concentrations of urea than of GuHCl. At neutral pH, the radius of the solvent-denatured state should be much smaller than for a random-flight chain and increase with either denaturant concentration or number of polar residues in the chain. A question of interest is whether free energies of folding should depend linearly on denaturant, as is often assumed. The free energy is predicted to be linear for urea but to have some small curvature for GuHCl. Predicted slopes and exposed areas of the unfolded states are found to be in generally good agreement with experiments. We also discuss stabilizing solvents and compare thermal with solvent denaturation.     

The nature of the free energy barriers to two-state folding

We introduce a simple procedure to analyze the temperature dependence of the folding and unfolding rates of two-state proteins. We start from the simple transition-state-like rate expression: k = D(eff)exp(-DeltaG(TS)/RT), in which upper and lower bounds for the intra-chain effective diffusion coefficient (D(eff)) are obtained empirically using the timescales of elementary processes in protein folding. From the changes in DeltaG(TS) as a function of temperature, we calculate enthalpies and heat capacities of activation, together with the more elusive entropies of activation. We then estimate the conformational entropy of the transition state by extrapolation to the temperature at which the solvation entropy vanishes by cancellation between polar and apolar terms. This approach is based on the convergence temperatures for the entropy of solvating apolar (approximately 385 K) and polar groups (approximately 335 K), the assumption that the structural properties of the transition state are somewhere in between the unfolded and folded states, and the established relationship between observed heat capacity and solvent accessibility.1 To circumvent the lack of structural information about transition states, we use the empirically determined heat capacities of activation as constraints to identify the extreme values of the transition state conformational entropy that are consistent with experiment. The application of this simple approach to six two-state folding proteins for which there is temperature-dependent data available in the literature provides important clues about protein folding. For these six proteins, we obtain an average equilibrium cost in conformational entropy of -4.3 cal x mol(-1)K(-1)per residue, which is in close agreement to previous empirical and computational estimates of the same quantity. Furthermore, we find that all these proteins have a conformationally diverse transition state, with more than half of the conformational entropy of the unfolded state. In agreement with predictions from theory and computer simulations, the transition state signals the change from a regime dominated by loss in conformational entropy to one driven by the gain in stabilization free energy (i.e., including protein interactions and solvation effects). Moreover, the height of the barrier is determined by how much stabilization free energy is realized at that point, which is related to the relative contribution of local versus non-local interactions. A remarkable observation is that the fraction of conformational entropy per residue that is present in the transition state is very similar for the six proteins in this study. Based on this commonality, we propose that the observed change in thermodynamic regime is connected to a change in the pattern of structure formation: from one driven by formation of pairwise interactions to one dominated by coupling of the networks of interactions involved in forming the protein core. In this framework, the barrier to two-state folding is crossed when the folding protein reaches a "critical native density" that allows expulsion of remaining interstitial water and consolidation of the core. The principle of critical native density should be general for all two-state proteins, but can accommodate different folding mechanisms depending on the particularities of the structure and sequence.     

Temperature dependence and thermodynamics of Klenow polymerase binding to primed-template DNA

DNA binding of Klenow polymerase has been characterized with respect to temperature to delineate the thermodynamic driving forces involved in the interaction of this polymerase with primed-template DNA. The temperature dependence of the binding affinity exhibits distinct curvature, with tightest binding at 25-30 degrees C. Nonlinear temperature dependence indicates Klenow binds different primed-template constructs with large heat capacity (DeltaCp) values (-870 to -1220 cal/mole K) and thus exhibits large temperature dependent changes in enthalpy and entropy. Binding is entropy driven at lower temperatures and enthalpy driven at physiological temperatures. Large negative DeltaCp values have been proposed to be a 'signature' of site-specific DNA binding, but type I DNA polymerases do not exhibit significant DNA sequence specificity. We suggest that the binding of Klenow to a specific DNA structure, the primed-template junction, results in a correlated thermodynamic profile that mirrors what is commonly seen for DNA sequence-specific binding proteins. Klenow joins a small number of other DNA-sequence independent DNA binding proteins which exhibit unexpectedly large negative DeltaCp values. Spectroscopic measurements show small conformational rearrangements of both the DNA and Klenow upon binding, and small angle x-ray scattering shows a global induced fit conformational compaction of the protein upon binding. Calculations from both crystal structure and solution structural data indicate that Klenow DNA binding is an exception to the often observed correlation between DeltaCp and changes in accessible surface area. In the case of Klenow, surface area burial can account for only about half of the DeltaCp of binding.     

Thermodynamic studies on the interaction between sodium n-dodecyl sulphate and histone H2B

The thermodynamic parameters for the interaction of the anionic detergent sodium n-dodecyl sulphate (SDS) with H2B at pH 3.2, 6.4 and 10 have been measured at 27 degrees C and 37 degrees C by equilibrium dialysis to determine the Gibbs energies of detergent binding. The data have been used to obtain the enthalpy of interaction from the temperature dependence of the equilibrium constants from the Van't Hoff relation. The enthalpy of interaction between H2B and SDS is endothermic at pH 3.2, 6.4 and 10. The shapes of the enthalpy curves at pH 3.2 and 10 show some small exothermic contribution which probably indicates folding of H2B. The interactions of H2B-SDS are dominated by the increase in entropy on detergent binding. The larger negative free energy, enthalpy and entropy changes at pH 6.4 are consistent with greater denaturation relative to pH 3.2 and 10.     

Thermodynamics of the temperature-induced unfolding of globular proteins.

The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature-induced unfolding of 11 globular proteins of known three-dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 degrees C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non-polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature-induced unfolding.     

Salt effects on the denaturation of DNA. IV. A calorimetric study of the helix-coil conversion of the alternating copolymer poly[d(A-T)].

The enthalpy deltaH, entropy deltaS, and the temperature Tm of the conformational transition of poly[d (A-T)] from the ordered to the randomly oriented state have been determined at pH 6.8 with the help of an adiabatic differential scanning calorimeter in Na2SO4 solutions of increasing ionic strength. Spectrophotometric denaturation experiments supplemented the calorimetric measurements. All thermodynamic parameters were found to vary strongly with salt concentration: both deltaH and Tm increase linearly with the logarithm of the mean molal activity alpha plus or minus of Na2SO4. However, whereas the dependence of Tm on salt activity remains linear over the entire salt concentration range employed deltaH decreases abruptly in the most concentrated Na2SO4 solutions. The entropy of melting changes with salt concentration in a pattern similar to that displayed by deltaH. The data on deltaH as well as data derived from the maximum slopes of the calorimetric heat denaturation curves were used to calculate the cooperative length Lh, the stacking free energy epsilon, and the cooperativity parameter sigma of poly[d(A-T)] as a function of ionic strength. Lh decreases with increasing salt concentration whereas sigma increases. Epsilon assumes more positive values with increasing salt molality. These changes then are in agreement with the generally held belief that an increase in salt concentration leads to an increase in the "loop" content of the copolymer.     

Muscarinic acetylcholine receptor: thermodynamic analysis of the interaction of agonists and antagonists

The thermodynamic parameters of the interaction of agonists and antagonists with heart and brain muscarinic receptors were determined. The binding of quinuclidinyl [3H]benzilate and the inhibition of quinuclidinyl benzilate (QNB) binding by agonists and antagonists were examined at temperatures between 2 degrees C and 27 degrees C. The density of specific binding sites and the relative proportions of high- and low-affinity binding components of drugs were unaffected by the temperature changes. The binding of atropine was entropy driven in brain and heart membranes. In contrast, net values of these thermodynamic parameters for QNB binding and for the high-affinity binding component of pirenzepine to brain membranes were decreased with the enhancement of the temperature. The low-affinity binding component of the agonists carbachol, oxotremorine and pilocarpine was enthalpy driven. Their high-affinity binding component was entropy driven at 2 degrees C and became enthalpy driven when the incubation temperature was increased. The guanine nucleotide Gpp[NH]p partly prevented the temperature-dependent decrease of net entropy and enthalpy values. Considering that the net changes of thermodynamic parameters are relevant of the interactions between the ligand, the receptor protein and the adjoining membranous molecules, a three-state conformational model is proposed for the muscarinic receptor protein. The receptor selectivity is reappreciated owing to these three states of the receptor protein and the different components of the muscarinic receptor complexes.     

Entropy increases from different sources support the high-affinity binding of the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases to the catalytic domains of matrix metalloproteinases-1 and -3

The avid binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is crucial for the regulation of pericellular and extracellular proteolysis. The interactions of the catalytic domain (cd) of MMP-1 with the inhibitory domains of TIMP-1 and TIMP-2 (N-TIMPs) and MMP-3cd with N-TIMP-2 have been characterized by isothermal titration calorimetry and compared with published data for the N-TIMP-1/MMP-3cd interaction. All interactions are largely driven by increases in entropy but there are significant differences in the profiles for the interactions of both N-TIMPs with MMP-1cd as compared with MMP-3cd; the enthalpy change ranges from small for MMP-1cd to highly unfavorable for MMP-3cd (-0.1 ± 0.7 versus 6.0 ± 0.5 kcal mol(-1)). The heat capacity change (ΔC(p)) of binding to MMP-1cd (temperature dependence of ΔH) is large and negative (-210 ± 20 cal K(-1) mol(-1)), indicating a large hydrophobic contribution, whereas the ΔC(p) values for the binding to MMP-3cd are much smaller (-53 ± 3 cal K(-1) mol(-1)), and some of the entropy increase may arise from increased conformational entropy. Apart from differences in ionization effects, it appears that the properties of the MMP may have a predominant influence in the thermodynamic profiles for these N-TIMP/MMP interactions.     

Interpretation of DSC data on protein denaturation complicated by kinetic and irreversible effects

Thermal denaturation of Kunitz soybean trypsin inhibitor (KTI) and ribulose-1,5-biphosphate carboxylase (RBPC) from tobacco leafs was studied by the method of high-sensitivity differential scanning calorimetry (HS-DSC). The dependence of the denaturation temperature on the heating rate reveals in the case of both proteins a non-equilibrium character of the denaturation transition in applied conditions. Developed kinetic approach allows the determination of an equilibrium transition temperature as well as the rate constants of denaturation and renaturation from the complex data of HS-DSC. This method was applied to the analysis of the pH-induced change of the conformational stability of KTI within pH range from 2.0 to 11.0. It allowed the determination of the pH dependencies: of the excess free energy of denaturation, of the activation enthalpy and entropy of denaturation as well as of the denaturation rate constant. Conclusions have been made suggesting the contribution of side-chain hydrogen bonds in the stabilisation of the native and activated states of KTI.     

Energetic basis of structural stability in the molten globule state: alpha-lactalbumin

The denatured states of alpha-lactalbumin, which have features of a molten globule state, have been studied to elucidate the energetics of the molten globule state and its contribution to the stability of the native conformation. Analysis of calorimetric and CD data shows that the heat capacity increment of alpha-lactalbumin denaturation highly correlates with the degree of disorder of the residual structure of the state. As a result, the denaturational transition of alpha-lactalbumin from the native to a highly ordered compact denatured state, and from the native to the disordered unfolded state are described by different thermodynamic functions. The enthalpy and entropy of the denaturation of alpha-lactalbumin to compact denatured state are always greater than the enthalpy and entropy of its unfolding. This difference represents the unfolding of the molten globule state. Calorimetric measurements of the heat effect associated with the unfolding of the molten globule state reveal that it is negative in sign over the temperature range of molten globule stability. This observation demonstrates the energetic specificity of the molten globule state, which, in contrast to a protein with unique tertiary structure, is stabilized by the dominance of negative entropy and enthalpy of hydration over the positive conformational entropy and enthalpy of internal interactions. It is concluded that at physiological temperatures the entropy of dehydration is the dominant factor providing stability for the compact intermediate state on the folding pathway, while for the stability of the native state, the conformational enthalpy is the dominant factor.     

Unifying temperature effects on the growth rate of bacteria and the stability of globular proteins

The specific growth rate constant for bacterial growth does not obey the Arrhenius-type kinetics displayed by simple chemical reactions. Instead, for bacteria, steep convex curves are observed on an Arrhenius plot at the low- and high-temperature ends of the biokinetic range, with a region towards the middle of the growth range loosely approximating linearity. This central region has been considered by microbiologists to be the "normal physiological range" for bacterial growth, a concept whose meaningfulness we now question. We employ a kinetic model incorporating thermodynamic terms for temperature-induced enzyme denaturation, central to which is a term to account for the large positive heat capacity change during unfolding of the proteins within the bacteria. It is now widely believed by biophysicists that denaturation of complex proteins and/or other macromolecules is due to hydrophobic hydration of non-polar compounds. Denaturation is seen as the process by which enthalpic and entropic forces becomes imbalanced both at high and at low temperatures resulting in conformational changes in the enzyme structure that expose hydrophobic amino acid groups to the surrounding water molecules. The "thermodynamic" rate model, incorporating the heat capacity change and its effect on the enthalpy and entropy of the system, fitted 35 sets of data for psychrophilic, psychrotrophic, mesophilic and thermophilic bacteria well, resulting in biologically meaningful estimates for the important thermodynamic parameters. As these results mirror those obtained by biophysicists for globular proteins, it appears that the same or a similar mechanism applies to bacteria as applies to proteins.     

The effects of polyols on the thermal stability of calf thymus DNA

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, (−) and (+) arabitol, -mannitol, -sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (Δ_dH) and temperature of the maximum of the denaturation peak (T_max) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of (−) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for (−) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of (−) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

Aromatic interactions in model systems

A thorough knowledge of noncovalent interactions is crucial to the understanding of biological complexity. One of the less well understood but significant weak interactions in nature is the aromatic interaction. Recent studies have provided new insight into the driving force, stability and selectivity of these interactions. The contribution of solvophobic and electrostatic interactions have been shown to be inextricably linked. Moreover, the influence of electrostatic and solvophobic components on the selectivity of aromatic interactions has been demonstrated.     

Solubility of proteins

A theory is presented on the solubility of proteins, in the hydrated as well as in the dry state, and in water as well as in organic solvents. To this effect, colloidal stability is assimilated with the solubility of the proteins, considered as hydrated entities. By means of a surface thermodynamic approach it can be shown that an increase in size of a hydrated protein must lead to insolubility, even in the absence of any change in a protein's surface properties. This can be substantiated experimentally by comparing the surface properties of immune complexes with those of their constituent immunoglobulins, as well as by comparing some of the properties of intact tobacco mosaic virus with those of its monomeric capsid subunits. Insolubilization of proteins by means of charge interactions as well as by dehydration is studied; an explanation is given of why precipitation caused by charge interactions is more likely to lead to partial irreversible denaturation than precipitation caused by protein-protein interactions brought about by partial dehydration (e.g., by salting-out). A link is established between the smallness (or even the negative value) of the interfacial tension between given proteins and various solvents and their solubility in these solvents. The energy of hydration of proteins can also be measured, and the differences between the free energies of interaction of dried and hydrated proteins with water point toward the additional processes underlying the solubilization, i.e., toward the conformational change of a protein in the process of becoming hydrated. The parameter of conformational change of a protein, while becoming hydrated, appears to be more closely linked to its degree of hydration than to its hydration energy.     

Temperature dependent mechanical unfolding and refolding of a protein studied by thermo-regulated optical tweezers

Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic contributions are obtained by quantifying the conformational equilibrium based on melting experiments performed in bulk conditions. Such experiments are suitable only for those small single-domain proteins whose side reactions of irreversible aggregation are unlikely to occur. Here, we avoid aggregation by pulling single-protein molecules in a thermo-regulated optical tweezers. Thus, we are able to explore the temperature dependence of the thermodynamic and kinetic parameters of MJ0366 from Methanocaldococcus jannaschii at the single-molecule level. By performing force-ramp experiments between 2°C and 40°C, we found that MJ0366 has a nonlinear dependence of free energy with temperature and a specific heat change of 2.3 ± 1.2 kcal/mol¹K. These thermodynamic parameters are compatible with a two-state unfolding/refolding mechanism for MJ0366. However, the kinetics measured as a function of the temperature show a complex behavior, suggesting a three-state folding mechanism comprising a high-energy intermediate state. The combination of two perturbations, temperature and force, reveals a high-energy species in the folding mechanism of MJ0366 not detected in force-ramp experiments at constant temperature.     

The osmophobic effect: natural selection of a thermodynamic force in protein folding

Intracellular organic osmolytes are present in certain organisms adapted to harsh environments and these osmolytes protect intracellular macromolecules against the denaturing environmental stress. In natural selection of organic osmolytes as protein stabilizers, it appears that the osmolyte property selected for is the unfavorable interaction between the osmolyte and the peptide backbone, a solvophobic thermodynamic force that we call the osmophobic effect. Because the peptide backbone is highly exposed to osmolyte in the denatured state, the osmophobic effect preferentially raises the free energy of the denatured state, shifting the equilibrium in favor of the native state. By focusing the solvophobic force on the denatured state, the native state is left free to function relatively unfettered by the presence of osmolyte. The osmophobic effect is a newly uncovered thermodynamic force in nature that complements the well-recognized hydrophobic interactions, hydrogen bonding, electrostatic and dispersion forces that drive protein folding. In organisms whose survival depends on the intracellular presence of osmolytes that can counteract denaturing stresses, the osmophobic effect is as fundamental to protein folding as these well-recognized forces.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.