Denaturational Heat Capacity Research Articles

Exothermic transitions in the heat capacity profiles of human cerebrospinal fluid.

In this work, we examined by DSC protein denaturation heat capacity profiles for two body fluids, cerebrospinal fluid (CSF) and blood plasma obtained from brain tumor (mainly glioblastoma) patients and healthy volunteers. We observed large distinctions between the heat capacity profiles of CSF and blood plasma, although their protein compositions are believed to have much in common. A prominent, previously unreported CSF feature was the existence of a pre-denaturation exothermic transition peaking at ~ 50-52°C, recorded for both control and brain tumor CSF. This appears to be the first observation of a pre-denaturation exotherm in a human body fluid. In all studied samples, the exotherms deconvoluted with high precision into a sum of two Gaussian peaks. These exotherms are apparently specific, originating from brain tissue-soluble proteins in the CSF not present in blood plasma. Malignant brain tumors (glioblastoma multiforme, Grade IV, and low-grade glioma, Grade II) reduced twofold the enthalpy of the exotherms relative to the control. These results suggest that the amount and/or conformational state of the CSF proteins (e.g., intrinsic disorder) giving rise to pre-denaturation exothermic events substantially changed upon brain tumor progression. Concomitantly, the enthalpy of the CSF endothermic peaks was partially redistributed from a lower-temperature (main) transition to a higher-temperature transition. The presented data demonstrated that the heat capacity profiles of intrinsic CSF proteins constitute a sensitive biomarker of glioblastoma and other brain malignancies.

Blood plasma thermal behavior and protein oxidation as indicators of multiple sclerosis clinical status and plasma exchange therapy progression

Multiple sclerosis (MS), a leading cause of physical disability in young adults, is a chronic autoimmune neuroinflammatory disease characterized by axonal injury and demyelination. In this study, denaturational heat capacity profiles of blood plasma from MS patients were recorded by DSC and used to monitor the effect of therapeutic plasma exchange. The results brought out certain, so far unknown correlations of the MS clinical status with the plasma heat capacity profiles and protein oxidation levels. Severe MS cases were accompanied by remarkably large deviations of the heat capacity profiles from the norm, manifested, in particular, in a strong suppression of the albumin peak. These deviations appear to result, at least partially, from plasma protein oxidation. Sequential plasma exchange procedures shifted plasma thermograms and protein oxidation levels towards norm, implying a beneficial plasma exchange effect. An indicator of the MS severity and plasma exchange effects based on the albumin denaturation enthalpy was introduced.

Relative stability of human centrins and its relationship to calcium binding.

Centrins are calcium binding proteins that belong to the EF-hand superfamily with diverse biological functions. Herein we present the first systematic study that establishes the relative stability of related centrins via complementary biophysical techniques. Our results define the stepwise molecular behavior of human centrins by two-dimensional infrared (2D IR) correlation spectroscopy, the change in heat capacity and enthalpy of denaturation by differential scanning calorimetry, and the relative stability of the helical regions of centrins by circular dichroism. More importantly, 2D IR correlation spectroscopy provides unique information about the similarities and differences in dynamics between these related proteins. The thermally induced molecular behavior of human centrins can be used to predict biological target interactions that have a relative dependence on calcium affinity. This information is essential for understanding why certain isoforms may be used to rescue a phenotype and therefore also for explaining the different functions these proteins may have in vivo. Furthermore, this comparative approach can be applied to the study of recombinant therapeutic protein candidates for the treatment of disease states.

Heightened stability of polcalcin Phl p 7 is correlated with strategic placement of apolar residues

Phl p 7 exhibits atypical conformational stability and a diminutive denaturational heat capacity increment, Δ C p . Because exposure of apolar surface largely dictates the magnitude of Δ C p , a depressed value could signify an unusually compact unfolded state. The volume of the denatured state ensemble (DSE) is evidently inversely correlated with mean hydrophobicity [Pace et al ., Protein Sci. 19 (2010) 929–943]. Interestingly, apolar residues replace more polar ones at four positions in Phl p 7. We herein examine the consequences of replacing those residues with the corresponding ones from Bra n 1, a related isoform. All four mutations – M4H, L21A, I60T, and C63A – destabilize Phl p 7. Our analysis suggests that the DSE of Phl p 7 is indeed highly compact and that the substitutions act by increasing its volume and solvent-accessibility. All four mutations increase the urea m value; L21A, I60T, and C63A also yield a perceptible increase in Δ C p .

Determination of heat capacity of unfolding for marginally stable proteins from a single temperature induced protein unfolding profile

A reliable estimation of heat capacity of denaturation (Δ C p ) is necessary to calculate the free energy of unfolding of proteins. For marginally stable proteins, such as mutants of a protein or proteins at low pH or under denaturating conditions, the pre-transition region is not fully populated by the native state. Analysis of differential scanning calorimeter (DSC) data under such conditions may not yield a reliable value of Δ C p and other associated thermodynamic parameters of unfolding. Analysis of denaturation profiles of (a) cytochrome c at pH 2.5, 3 and 8 and (b) myoglobin at pH 4, show that an accurate value of Δ C p can be extracted from a single unfolding profile obtained spectroscopically by including low temperature data.

Polcalcin Divalent Ion-Binding Behavior and Thermal Stability: Comparison of Bet v 4, Bra n 1, and Bra n 2 to Phl p 7

Polcalcins are pollen-specific proteins containing two EF-hands. Atypically, the C-terminal EF-hand binding loop in Phl p 7 (from timothy grass) harbors five, rather than four, anionic side chains, due to replacement of the consensus serine at -x by aspartate. This arrangement has been shown to heighten parvalbumin Ca(2+) affinity. To determine whether Phl p 7 likewise exhibits anomalous divalent ion affinity, we have also characterized Bra n 1 and Bra n 2 (both from rapeseed) and Bet v 4 (from birch tree). Relative to Phl p 7, they exhibit N-terminal extensions of one, five, and seven residues, respectively. Interestingly, the divalent ion affinity of Phl p 7 is unexceptional. For example, at -17.84 +/- 0.13 kcal mol(-1), the overall standard free energy for Ca(2+) binding falls within the range observed for the other three proteins (-17.30 +/- 0.10 to -18.15 +/- 0.10 kcal mol(-1)). In further contrast to parvalbumin, replacement of the -x aspartate, via the D55S mutation, actually increases the overall Ca(2+) affinity of Phl p 7, to -18.17 +/- 0.13 kcal mol(-1). Ca(2+)-free Phl p 7 exhibits uncharacteristic thermal stability. Whereas wild-type Phl p 7 and the D55S variant denature at 77.3 and 78.0 degrees C, respectively, the other three polcalcins unfold between 56.1 and 57.9 degrees C. This stability reflects a low denaturational heat capacity increment. Phl p 7 and Phl p 7 D55S exhibit DeltaC(p) values of 0.34 and 0.32 kcal mol(-1) K(-1), respectively. The corresponding values for the other three polcalcins range from 0.66 to 0.95 kcal mol(-1) K(-1).

Solid state chemistry of proteins IV. what is the meaning of thermal denaturation in freeze dried proteins?

This research addresses the thermodynamic significance of the denaturation endotherm observed during differential scanning calorimetry (DSC) scans of proteins in dry formulations, such as freeze dried solids. Human growth hormone formulations are the chosen representative examples. We employ observations of denaturation temperature, glass transition temperature, and the differences between estimated molecular mobilities to argue that unfolding is under partial thermodynamic control. Further, unfolding during a DSC scan is simulated using a three state kinetic model, which is a two state unfolding model followed by aggregation. Kramers-type rate constants are used, where the preexponential term is dominated by viscous forces. Simulation results are in qualitative agreement with experiment, and clearly show that while the denaturation endotherm is impacted by irreversibility, caused by nonzero scan rate and aggregation, the position of the endotherm peak is changed only slightly. Thus, the denaturation peak is a good approximation for the thermodynamic denaturation temperature. Using data for denaturation temperature, heat of denaturation, and heat capacity of denaturation, free energy versus temperature curves were calculated. We find that even formulations with added saccharides are thermodynamically unstable near ambient temperature; significant denaturation in the solid state is prevented by low mobility.

Is there a relationship between protein thermal stability and the denaturation heat capacity change?

It is shown the non-occurrence of a correlation between the values of the denaturation temperature and those of the denaturation heat capacity change for a set of 13 proteins possessing the ‘SH3-type’ fold from both mesophilic and thermophilic microorganisms. This seems to be a rather general result, because, fixed the size and the folding pattern, the denaturation heat capacity change is a nearly constant quantity, within the uncertainty limits of experimental determinations, regardless of the thermal stability of the protein. A precise definition of the thermodynamics of the hydrophobic effect is presented to clarify that the above finding does not imply that the hydrophobic effect does not play a role in the extra-thermal stability of thermophilic and hyperthermophilic proteins.

Pneumococcal phosphorylcholine esterase, Pce, contains a metal binuclear center that is essential for substrate binding and catalysis

The phosphorylcholine esterase from Streptococcus pneumoniae, Pce, catalyzes the hydrolysis of phosphorylcholine residues from teichoic and lipoteichoic acids attached to the bacterial envelope and comprises a globular N-terminal catalytic module containing a zinc binuclear center and an elongated C-terminal choline-binding module. The dependence of Pce activity on the metal/enzyme stoichiometry shows that the two equivalents of zinc are essential for the catalysis, and stabilize the catalytic module through a complex metal-ligand coordination network. The pH dependence of Pce activity toward the alternative substrate p-nitrophenylphosphorylcholine (NPPC) shows that k(cat) and k(cat)/K(m) depend on the protonation state of two protein residues that can be tentatively assigned to the ionization of the metal-bound water (hydrogen bonded to D89) and to H228. Maximum activity requires deprotonation of both groups, although the catalytic efficiency is optimum for the single deprotonated form. The drastic reduction of activity in the H90A mutant, which still binds two Zn2+ ions at neutral pH, indicates that Pce activity also depends on the geometry of the metallic cluster. The denaturation heat capacity profile of Pce exhibits two peaks with T(m) values of 39.6 degrees C (choline-binding module) and 60.8 degrees C (catalytic module). The H90A mutation reduces the high-temperature peak by about 10 degrees C. Pce is inhibited in the presence of 1 mM zinc, but this inhibition depends on pH, buffer, and substrate species. A reaction mechanism is proposed on the basis of kinetic data, the structural model of the Pce:NPPC complex, and the currently accepted mechanism for other Zn-metallophosphoesterases.

Stabilization Mechanism of the Tryptophan Synthase α-Subunit from Thermus thermophilus HB8: X-Ray Crystallographic Analysis and Calorimetry

In order to elucidate the thermo-stabilization mechanism of the tryptophan synthase alpha-subunit from the extreme thermophile Thermus thermophilus HB8 (Tt-alpha-subunit), its crystal structure was determined and its stability was examined using DSC. The results were compared to those of other orthologs from mesophilic and hyperthermophilic organisms. The denaturation temperature of the Tt-alpha-subunit was higher than that of the alpha-subunit from S. typhimurium (St-alpha-subunit) but lower than that of the alpha-subunit from P. furiosus (Pf-alpha-subunit). Specific denaturation enthalpy and specific denaturation heat capacity values of the Tt-alpha-subunit were the lowest among the three proteins, suggesting that entropy effects are responsible for the stabilization of the Tt-alpha-subunit. Based on a structural comparison with the St-alpha-subunit, two deletions in loop regions, an increase in the number of ion pairs and a decrease in cavity volume seem to be responsible for the stabilization of the Tt-alpha-subunit. The results of structural comparison suggest that the native structure of the Tt-alpha-subunit is better adapted to an ideally stable structure than that of the St-alpha-subunit, but worse than that of the Pf-alpha-subunit. The results of calorimetry suggest that the residual structure of the Tt-alpha-subunit in the denatured state contributes to the stabilization.

Why Is Trehalose an Exceptional Protein Stabilizer?: AN ANALYSIS OF THE THERMAL STABILITY OF PROTEINS IN THE PRESENCE OF THE COMPATIBLE OSMOLYTE TREHALOSE

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.

Relationships between the temperature dependence of solvent denaturation and the denaturant dependence of protein stability curves

We have used a simple binding model to consider how the thermodynamics of denaturant–protein interactions might influence the shape of protein stability curves (free energy change as a function of temperature), and how these effects translate into a temperature dependence of the apparent m-value (sensitivity of unfolding free energy to denaturant). We find that for an exothermic binding reaction with no binding heat capacity increment, increasing denaturant concentrations produces an apparent increase in curvature in the protein stability curve, giving rise to an increase in the heat capacity increment of unfolding. Similar increases are seen if the binding heat capacity increment is taken as positive. However, for a negative binding heat capacity increment, increasing denaturant concentrations decreases the curvature of the stability curve, giving rise to a decrease in the heat capacity of unfolding. At very high denaturant concentrations (above which the heat capacity of denaturation becomes negative) the stability curve becomes dimpled, showing two separate maxima rather than one. These three models result in very different temperature dependencies of apparent m-values. For urea-induced unfolding of the ankyrin-domain of the Drosophila Notch protein, we find a dependence of experimental m-values on temperature that is similar to that produced by a negative binding heat capacity increment. This temperature dependence is consistent with the observed decrease in heat capacity of unfolding as denaturant is added.

Effects of some metal ions on the denaturational heat capacity increments in dilute solutions of ds-DNA

The native DNA duplex may be viewed as a cooperatively-ordered H bonded structure, including well localized Watson–Crick base pairs and H-bounded networks of hydration parts of the DNA–solvent interface in the grooves of the helix. In this paper, we present the effect of different metal ions (Li +, Mg 2+ and Cu 2+) on the denaturational heat capacity increment (Δ C p ) for calf thymus DNA. Since the contribution from the ordered hydration water fraction disruption energy to the total enthalpy and heat capacity increment values of double helix melting is significant, it must be possible to detect the effect of metal ions on the structural ordering/disordering of the H-bounded network in the hydration shell of the DNA duplex. Experimental results suggest that Li +, which is preferentially adsorbed in the minor groove of B-DNA and should contribute significantly to the stabilization of B-form, has a pronounced influence on the value of Δ C p . For the system Mg 2+–DNA, the values of Δ C p are also significant, which can be explained by the formation of inner hydration sphere complexes and immobilization of structural water by the grooves of the duplex, stabilizing the helix. The effect of Cu 2+ ions is much more pronounced. The satellite peaks of calf thymus DNA become lower as the concentration of Cu 2+ increases and for concentrations of Cu 2+ higher than 0.010 M, they disappear and Δ C p =0. This is indicative of the known preference of Cu 2+ for purins and GC rich sites of DNA, binding to the N7 of guanine. As a result, disruption of the base stacking and hydrogen bounded water networks in the grooves of the double helix takes place.

Comparative analyses of the conformational stability of a hyperthermophilic protein and its mesophilic counterpart

Comparison of the conformational stability of an O(6)-methylguanine-DNA methyltransferase (MGMT) from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-MGMT), and its mesophilic counterpart C-terminal Ada protein from Escherichia coli (Ec-AdaC) was performed in order to obtain information about the relationship between thermal stability and other factors, such as thermodynamic parameters, thermodynamic stability and other unfolding conditions. Tk-MGMT unfolded at Tm = 98.6 degrees C, which was 54.8 degrees C higher than the unfolding temperature of Ec-AdaC. The maximum free energy (DeltaG(max)) of the proteins were different; the value of Tk-MGMT (42.9 kJ.mol-1 at 29.5 degrees C) was 2.6 times higher than that of Ec-AdaC (16.6 kJ.mol-1 at 7.4 degrees C). The high conformational stability of Tk-MGMT was attributed to a 1.6-fold higher enthalpy value than that of Ec-AdaC. In addition, the DeltaG(max) temperature of Tk-MGMT was considerably higher (by 22.1 degrees C). The apparent heat capacity of denaturation (DeltaC(p)) of Tk-MGMT was 0.7-fold lower than that of Ec-AdaC. These three synergistic effects, increasing DeltaGmax, shifted DeltaG vs. temperature curve, and low DeltaC(p), give Tk-MGMT its thermal stability. Unfolding profiles of the two proteins, tested with four alcohols and three denaturants, showed that Tk-MGMT possessed higher stability than Ec-AdaC in all conditions studied. These results indicate that the high stability of Tk-MGMT gives resistance to chemical unfolding, in addition to thermal unfolding.

On the stabilizing action of protein denaturants: acetonitrile effect on stability of lysozyme in aqueous solutions

Stability of hen lysozyme in the presence of acetonitrile (MeCN) at different pH values of the medium was studied by scanning microcalorimetry with a special emphasis on determination of reliable values of the denaturational heat capacity change. It was found that the temperature of denaturation decreases on addition of MeCN. However, the free energy extrapolation showed that below room temperature the thermodynamic stability increases at low concentrations of MeCN in spite of the general destabilizing effect at higher concentrations and temperatures. Charge-induced contribution to this stabilization was shown to be negligible (no pH-dependence was found); therefore, the most probable cause for the phenomenon is an increase of hydrophobic interactions at low temperatures in aqueous solutions containing small amounts of the organic additive. The difference in preferential solvation of native and denatured states of lysozyme was calculated from the stabilization free energy data. It was found that the change in preferential solvation strongly depends on the temperature in the water-rich region. At the higher MeCN content this dependence decreases until, at 0.06 mole fractions of MeCN, the difference in the preferential solvation between native and denatured lysozyme becomes independent of the temperature over a range of 60 K. The importance of taking into account non-ideality of a mixed solution, when analyzing preferential solvation phenomena was emphasized.

Heat capacity and compactness of denatured proteins

One of the striking results of protein thermodynamics is that the heat capacity change upon denaturation is large and positive. This change is generally ascribed to the exposure of non-polar groups to water on denaturation, in analogy to the large heat capacity change for the transfer of small non-polar molecules from hydrocarbons to water. Calculations of the heat capacity based on the exposed surface area of the completely unfolded denatured state give good agreement with experimental data. This result is difficult to reconcile with evidence that the heat denatured state in the absence of denaturants is reasonably compact. In this work, sample conformations for the denatured state of truncated CI2 are obtained by use of an effective energy function for proteins in solution. The energy function gives denatured conformations that are compact with radii of gyration that are slightly larger than that of the native state. The model is used to estimate the heat capacity, as well as that of the native state, at 300 and 350 K via finite enthalpy differences. The calculations show that the heat capacity of denaturation can have large positive contributions from non-covalent intraprotein interactions because these interactions change more with temperature in non-native conformations than in the native state. Including this contribution, which has been neglected in empirical surface area models, leads to heat capacities of unfolding for compact denatured states that are consistent with the experimental heat capacity data. Estimates of the stability curve of CI2 made with the effective energy function support the present model.

Prediction of the heat capacity change on thermal denaturation of globular proteins

The large positive heat capacity change is a common feature of both the transfer of nonpolar compounds to water and the temperature-induced denaturation of globular proteins. In this paper we present a model for the calculation of the denaturation heat capacity change, that is a key parameter of protein thermodynamics, by means of a simple group additivity scheme and some common structural properties of globular proteins. The specific polar and nonpolar contributions to the heat capacity change are determined by analyzing the transfer of several different series of organic compounds to water. Additionally, we derive a general relationship for the evaluation of the fraction of accessible surface area buried in the protein interior, whose knowledge is necessary because only the groups that contact water on unfolding contribute to the heat capacity change. The model, despite its simplicity, works well, as the agreement between the calculated and experimental values of the denaturation heat capacity change is satisfactory for a large set of globular proteins.

Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus.

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.

Heat capacity and thermodynamic characteristics of denaturation and glass transition of hydrated and anhydrous proteins

Calorimetric measurements of absolute heat capacity have been performed for hydrated 11S-globulin (0 < C H 2O < 25%) and for lysozyme in a concentrated solution, both in the native and denatured states. The denaturation process is observed in hydrated and completely anhydrous proteins; it is accompanied by the appearance of heat capacity increment ( Δ N D C p), as is the case for protein solutions. It has been shown that, depending on the temperature and water content, the hydrated denatured proteins can be in a highly elastic or glassy states. Glass transition is also observed in hydrated native proteins. It is found that the denaturation increment Δ N D C p in native protein, like the increment ΔC p in denatured protein in glass transition at low water contents, is due to additional degrees of freedom of thermal motion in the protein globule. In contrast to the conventional notion, comparison of absolute C p values for hydrated denatured proteins with the C p values for denatured proteins in solution has indicated a dominant contribution of the globule thermal motion to the denaturation increment of protein heat capacity in solutions. The concentration dependence of denaturing heat absorption (temperature at its maximum, T D, and thermal effect, ΔQ D) and that of glass transition temperature, T g, for 11S-globulin have been studied in a wide range of water contents. General polymeric and specific protein features of these dependencies are discussed.

An alternative approach in the structure-based predictions of the thermodynamics of protein unfolding

A new approach for a first-order prediction of the thermodynamic properties of small globular proteins has been developed. The method put forward here has been shown to be successful in predicting, within acceptable margins of uncertainty, the denaturational heat capacity changes of a given protein if its amino acid composition is known. If compared with other models this method has the following advantages: (1) no details about the three-dimensional structure of the protein are required; (2) comparison with the thermodynamic properties of small model compounds is not necessary: (3) the temperature dependence of the denaturational heat capacity change is taken into account. Moreover, the equations developed have allowed us to point out the errors that can be made if the temperature-dependence of the denaturational heat capacity change is not taken into account in the calculation of the unfolding thermodynamic functions.