Identification of pitfalls in the analysis of heat capacity changes of beta-lactoglobulin A

Information on changes in heat capacity (DeltaCp) of proteins upon unfolding is used frequently in literature to understand possible follow-up reactions of protein denaturation, like their aggregation propensity. This thermodynamic property is intrinsic to the protein's architecture and unfolding and should be independent of the approach used to evaluate it. However, for many proteins, the reported values for DeltaCp vary considerably. To identify whether the origin of these discrepancies lies within the experimental approach chosen and/or in the too simplified unfolding models used in the analysis of the data, we choose beta-lactoglobulin A, a relatively small protein, but disputed for its two-state unfolding, and established its DeltaCp from tryptophan fluorescence, near-UV circular dichroism and differential scanning calorimetric measurements. In view of the large variation for the obtained DeltaCp (between 3.2 and 10.1+/-0.8 kJ/(mol K)), it is evident that: (1) the sensitivity of different approaches to the structural changes; (2) irreversibility of unfolding; (3) non-ideal two-state unfolding behaviour need to be considered prior to interpretation. While the first two points can be addressed by using multiple approaches, the applicability of the selected unfolding behaviour for the analysis is often less easy to establish. In this work, we illustrate that by checking the wavelength-dependence used to detect protein conformational changes a tool is provided that gives a direct insight in the validity of the interpretation in these studies. An experimentally validated determination of DeltaCp allows a more proper use for the mechanistic understanding of protein denaturation and its follow-up reactions, avoiding pitfalls in the interpretation.     

Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects

Using the heat capacity values for amino acid side-chains and the peptide unit determined in the accompanying paper, we calculated the partial heat capacities of the unfolded state for four proteins (apomyoglobin, apocytochrome c, ribonuclease A, lysozyme) in aqueous solution in the temperature range from 5 to 125 degrees C, with an assumption that the constituent amino acid residues contribute additively to the integral heat capacity of a polypeptide chain. These ideal heat capacity functions of the extended polypeptide chains were compared with the calorimetrically determined heat capacity functions of the heat and acid-denatured proteins. The average deviation of the experimental functions from the calculated ideal ones in the whole studied temperature range does not exceed the experimental error (5%). Therefore, the heat-denatured state of a protein, in solutions with acidic pH preventing aggregation, approximates well the completely unfolded state of this macromolecule. The heat capacity change caused by hydration of amino acid residues upon protein unfolding was also determined and it was shown that this is the major contributor to the observed heat capacity effect of unfolding. Its value is different for different proteins and correlates well with the surface area of non-polar groups exposed upon unfolding. The heat capacity effect due to the configurational freedom gain by the polypeptide chain was found to contribute only a small part of the overall heat capacity change on unfolding.     

Analysis of the heat capacity dependence of protein folding.

This paper presents an analysis of plots of enthalpy versus heat capacity change at 25 degrees C for the unfolding of proteins and for the dissolution of gaseous, liquid and solid solutes, first reported by Murphy, Privalov & Gill. The negative slope in the enthalpy plot for proteins is interpreted as arising from a large penalty associated with burying polar groups in the protein interior. The small enthalpy changes that accompany protein unfolding at 25 degrees C are also discussed. It is argued that the combined effects of hydrogen bond formation and close packing predict a large positive enthalpy of unfolding. Electrostatic calculations indicate that the penalty associated with burying polar groups is large enough to effectively cancel these terms, leading to the small net enthalpy changes that are observed. The free energy changes associated with protein folding are also discussed. The free energy cost of burying polar groups largely compensates for the stabilizing contribution of the hydrophobic effect and would appear to account for the fact that proteins are marginally stable, independent of their size and of their relative hydrophobicities.     

The heat capacity of proteins

The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25°C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25°C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to ～5%). It is demonstrated that a single universal mathematical function can be used to represent the partial molar heat capacity of the native and unfolded states of proteins in solution. This function can be experimentally written in terms of the molecular weight, the polar and apolar solvent accessible surface areas, and the total area buried from the solvent. This unique function accurately predicts the different magnitude and temperature dependences of the heat capacity of both the native and unfolded states, and therefore of the heat capacity changes associated with folding/unfolding transitions. © 1995 Wiley-Liss, Inc.     

Thermodynamics of unfolding of the alpha-amylase inhibitor tendamistat. Correlations between accessible surface area and heat capacity.

Unfolding of the small alpha-amylase inhibitor tendamistat (74 residues, 2 disulfide bridges) has been characterized thermodynamically by high sensitivity scanning microcalorimetry. To link the stability parameters with structural information we use heat capacity group parameters and water accessible surface areas to calculate the change in heat capacity on unfolding of tendamistat. Our results show that both the group parameter and surface area approaches provide a reasonable, though not perfect, basis for delta Cp calculations. When using the experimentally determined temperature-independent heat capacity increase of 2.89 kJ mol-1 K-1 tendamistat exhibits convergence of thermodynamic parameters at about 140 degrees C, in agreement with recent predictions of the temperature at which the hydrophobic hydration is supposed to disappear. Despite the apparent support of this new view of the hydrophobic effect, there are inconsistencies in the interpretation of the thermodynamic parameters and these are addressed in the Discussion. The specific stability of tendamistat is similar to that of modified bovine pancreatic trypsin inhibitor, with only two of the native three disulfide bridges intact. This observation confirms our previous conclusion that disulfide bridges affect significantly the enthalpy and entropy of unfolding. The recent study by Doig & Williams provides additional convincing support for this conclusion. The predictive scheme proposed by these authors permits a fair estimate of the Gibbs free energy and enthalpy changes of these two proteins.     

Two types of arrestins expressed in medaka rod photoreceptors

Globular protein thermostability is characterized the cold denaturation, maximal stability (Tms) and heat denaturation temperatures. For mesophilic globular proteins, Tms typically ranges from -25 degrees C to +35 degrees C. We show that the indirect estimate of Tms from calorimetry and the direct estimate from chemical denaturation performed in a range of temperatures are in close agreement. The heat capacity change of unfolding per mol residue (delta Cp) alone is shown to accurately predict Tms. Delta Cp and hence Tms can be predicted solely from the protein sequence. The average difference in free energy of unfolding at the observed and predicted values of Tms is 1.0 kcal mol(-1), which is small compared to typical values of the total free energy of unfolding.     

Thermodynamics of unfolding of an integral membrane protein in mixed micelles

Quantitative studies of membrane protein folding and unfolding can be difficult because of difficulties with efficient refolding as well as a pronounced propensity to aggregate. However, mixed micelles, consisting of the anionic detergent sodium dodecyl sulfate and the nonionic detergent dodecyl maltoside facilitate reversible and quantitative unfolding and refolding. The 4-transmembrane helix protein DsbB from the inner membrane of Escherichia coli unfolds in mixed micelles according to a three-state mechanism involving an unfolding intermediate I. The temperature dependence of the kinetics of this reaction between 15 degrees and 45 degrees C supports that unfolding from I to the denatured state D is accompanied by a significant decrease in heat capacity. For water-soluble proteins, the heat capacity increases upon unfolding, and this is generally interpreted as the increased binding of water to the protein as it unfolds, exposing more surface area. The decrease in DsbB's heat capacity upon unfolding is confirmed by independent thermal scans. The decrease in heat capacity is not an artifact of the use of mixed micelles, since the water soluble protein S6 shows conventional heat-capacity changes in detergent. We speculate that it reflects the binding of SDS to parts of DsbB that are solvent-exposed in the native DM-bound state. This implies that the periplasmic loops of DsbB are relatively unstructured. This anomalous thermodynamic behavior has not been observed for beta-barrel membrane proteins, probably because they do not bind SDS so extensively. Thus the thermodynamic behavior of membrane proteins appears to be intimately connected to their detergent-binding properties.     

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.     

Contribution of hydration and non-covalent interactions to the heat capacity effect on protein unfolding.

The heat capacity change upon protein unfolding has been analysed using the heat capacity data for the model compounds' transfer into water, corrected for volume effects. It has been shown that in the unfolding, the heat capacity increment is contributed to by the effect of hydration of the non-polar groups, which is positive and decreases with temperature increase, and by the effect of hydration of the polar groups, which is negative and decreases in magnitude as temperature increases. The sum of these two effects is very close to the total heat capacity increment of protein unfolding at room temperature but is likely to deviate from it at higher temperatures. Therefore, the expected heat capacity effect caused by the increase of configurational freedom of the polypeptide chain upon unfolding seems to be compensated for by some other effect, perhaps associated with fluctuation of the native protein structure.     

Structural energetics of barstar studied by differential scanning microcalorimetry.

The energetics of barstar denaturation have been studied by CD and scanning microcalorimetry in an extended range of pH and salt concentration. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two-state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at approximately 75 degrees C was found to be 5.6 +/- 0.3 kJ K-1 mol-1. In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to explain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration were found to be in good agreement with those calculated for other proteins, but the enthalpy and entropy of breaking internal interactions were found to be among the lowest for all globular proteins that have been studied. Additionally, the partial specific heat capacity of barstar in the native state was found to be 0.37 +/- 0.03 cal K-1 g-1, which is higher than what is observed for most globular proteins and suggests significant flexibility in the native state. It is known from structural data that barstar undergoes a conformational change upon binding to its natural substrate barnase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal unfolding and aggregation of actin

Actin is one of the most abundant proteins in nature. It is found in all eukaryotes and plays a fundamental role in many diverse and dynamic cellular processes. Also, actin is one of the most ubiquitous proteins because actin-like proteins have recently been identified in bacteria. Actin filament (F-actin) is a highly dynamic structure that can exist in different conformational states, and transitions between these states may be important in cytoskeletal dynamics and cell motility. These transitions can be modulated by various factors causing the stabilization or destabilization of actin filaments. In this review, we look at actin stabilization and destabilization as expressed by changes in the thermal stability of actin; specifically, we summarize and analyze the existing data on the thermal unfolding of actin as measured by differential scanning calorimetry. We also analyze in vitro data on the heat-induced aggregation of actin, the process that normally accompanies actin thermal denaturation. In this respect, we focus on the effects of small heat shock proteins, which can prevent the aggregation of thermally denatured actin with no effect on actin thermal unfolding. As a result, we have proposed a mechanism describing the thermal denaturation and aggregation of F-actin. This mechanism explains some of the special features of the thermal unfolding of actin filaments, including the effects of their stabilization and destabilization; it can also explain how small heat shock proteins protect the actin cytoskeleton from damage caused by the accumulation of large insoluble aggregates under heat shock conditions.     

Conformational stability of legume lectins reflect their different modes of quaternary association: solvent denaturation studies on concanavalin A and winged bean acidic agglutinin

Thermodynamic parameters associated with the unfolding of the legume lectin, WBA II, were determined by isothermal denaturation. The analysis of isothermal denaturation data provided values for conformational stability and heat capacity for WBA II unfolding. To explore the role of intersubunit contact in stability, we carried out similar studies under identical conditions on Concanavalin A, a legume lectin of nearly similar size, buried hydrophobic surface area and tertiary structure to that of WBA II but with a different oligomerization pattern. Both proteins showed a reversible two-state unfolding with guanidine hydrochloride. As expected, the change in heat capacity upon unfolding was similar for both proteins at 3.5 and 3.7 kcal mol(-1) K(-1) for Concanavalin A and WBA II, respectively. Although the deltaG(H20) at the maximum stability of both proteins is around 16 kcal/mol, Concanavalin A exhibits greater stability at higher temperatures. The T(g) obtained for Concanavalin A and WBA II were 21 degrees C apart at 87.2 and 66.6 degrees C, respectively. The higher conformational stability at higher temperatures and the T(g) of Concanavalin A as compared to that of WBA II are largely due to substantial differences in the degree of subunit contact in these dimeric proteins. Ionic interactions and hydrogen bonding between the monomers of the two proteins also seem to play a significant role in the observed stability differences between these two proteins.     

A thermodynamic comparison of mesophilic and thermophilic ribonucleases H

The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues. To gain insight into the basis of thermostability, we have determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins. Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function.     

Stability of recombinant Lys25-ribonuclease T1

The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn.HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees C: DSC, 46.6 +/- 2.1 kJ/mol; UV melting curves, 48.7 +/- 5 kJ/mol; Gdn.HCl transition curves, 40.8 +/- 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van't Hoff and calorimetric enthalpy, delta HvH/delta Hcal, is 0.97 +/- 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by delta Cp degrees = 5.1 +/- 0.5 kJ/(mol.K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric study of tryptophan synthase alpha-subunit and two mutant proteins

Heat-denaturation of tryptophan synthase alpha-subunit from E. coli and two mutant proteins (Glu 49 leads to Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20 degrees, Cp20, has been found to be (0.43 +/- 0.02) cal . k-1 . g-1, the unfolding heat capacity change, delta dCp, (0.10 +/- 0.01) cal . K-1 . g-1, and the unfolding enthalpy value extrapolated to 110 degrees, delta dh110, (9.3 +/- 0.5) cal . g-1 for the three proteins. The value of Cp20 was larger than those found for "fully compact protein" and that of delta dh110 was smaller. Unfolding Gibbs energy, delta dG at 25 degrees for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal . mol-1 at pH 9.3, respectively. Unfolding enthalpy, delta dH, of the three proteins seemed to be the same and equal to (23.2 +/- 1.2) kcal . mol-1 at 25 degrees. As a consequence of the same value of delta dH and the different value in delta dG, substantial differences in unfolding entropy, delta dS, were found for the three proteins. The values of delta dG for the three proteins at 25 degrees coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.     

Protein stability curves

The stability curve of a protein is defined as the plot of the free energy of unfolding as a function of temperature. For most proteins the change in heat capacity on denaturation, or unfolding, is large but approximately constant. When unfolding is s two-state process, most of the salient features of the stability curves of proteins can be derived from this fact. A number of relations are obtained, including the special features of low-temperature denaturation, the properties of the maximum in stability, and the interrelationships of the characteristic temperatures of the protein. The paper closes with a formula that permits one to calculate small changes in stabilization free energy from changes in the melting temperature of the protein.     

Chemical modification of tryptophan residues and stability changes in proteins

The role of tryptophan residues in the stability of proteins was studied by ozone oxidation, which causes a small change in the tryptophan side chain. Trp 187 of the constant fragment of a type lambda immunoglobulin light chain, Trp 59 of ribonuclease T1, and Trp 62 of hen egg white lysozyme were oxidized specifically by ozone to N'-formylkynurenine or kynurenine. Judging from their circular dichroic and fluorescence spectra, these modified proteins were found to be the same as those of the respective intact proteins. However, even the slight modification of a single tryptophan residue produced a large decrease in the stability of these proteins to guanidine hydrochloride and heat. The smaller the extent of exposure of the tryptophan residue, the greater the effect of the modification on the stability. The formal kinetic mechanism of unfolding and refolding by guanidine hydrochloride of the CL fragment was not altered by tryptophan oxidation, but the rate constants for unfolding and refolding changed. The thermal unfolding transitions were analyzed to obtain the thermodynamic parameters. The enthalpy and entropy changes for the modified proteins were larger than the respective values for the intact proteins.     

Beyond transcription--new mechanisms for the regulation of molecular chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions-the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

Napin from Brassica juncea: thermodynamic and structural analysis of stability

The napin from Brassica juncea, oriental mustard, is highly thermostable, proteolysis resistant and allergenic in nature. It consists of two subunits - one small (29 amino acid residues) and one large (86 amino acids residues) - held together by disulfide bonds. The thermal unfolding of napin has been followed by differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. The thermal unfolding is characterized by a three state transition with T(M1) and T(M2) at 323.5 K and 335.8 K, respectively; DeltaC(P1) and DeltaC(P2) are 2.05 kcal mol(-1) K(-1) and 1.40 kcal mol(-1) K(-1), respectively. In the temperature range 310-318 K, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N <_-_-> I <_-_-> U with DeltaG(1H2O) and DeltaG(2H2O) values of 5.2 kcal mol(-1) and 5.1 kcal mol(-1) at 300 K, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from 20 A to 35.0 A due to unfolding by guanidinium hydrochloride. In silico alignment of sequences of napin has revealed that the internal repeats (40%) spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species. The internal repeats may contribute to the greater stability of napin. A thorough understanding of the structure and stability of these proteins is essential before they can be exploited for genetic improvements for nutrition.     

Multifrequency calorimetry of the folding/unfolding transition of cytochrome c.

The folding-unfolding transition of Fe(III) cytochrome c has been studied with the new technique of multifrequency calorimetry. Multifrequency calorimetry is aimed at measuring directly the dynamics of the energetic events that take place during a thermally induced transition by measuring the frequency dispersion of the heat capacity. This is done by modulating the folding/unfolding equilibrium using a variable frequency, small oscillatory temperature perturbation (approximately 0.05-0.1 degrees C) centered at the equilibrium temperature of the system. Fe(III) cytochrome c at pH 4 undergoes a fully reversible folding/unfolding transition centered at 67.7 degrees C and characterized by an enthalpy change of 81 kcal/mol and heat capacity difference between unfolded and folded states of 0.9 kcal/K*mol. By measuring the temperature dependence of the frequency dispersion of the heat capacity in the frequency range of 0.1-1 Hz it has been possible to examine the time regime of the enthalpic events associated with the transition. The multifrequency calorimetry results indicate that approximately 85% of the excess heat capacity associated with the folding/unfolding transition relaxes with a single relaxation time of 326 +/- 68 ms at the midpoint of the transition region. This is the first time that the time regime in which heat is absorbed and released during protein folding/unfolding has been measured.