Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments

Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.     

Heat capacity changes upon burial of polar and nonpolar groups in proteins

In this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, DeltaC(p), that have an opposite sign, negative for nonpolar groups and positive for polar groups. To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41). We substituted Val at positions 5 and 17 and Leu at position 67 with a polar residue, Asn. As a control, Ala was introduced at the same three positions. We also replaced the buried polar Gln41 with Val and Leu, nonpolar residues that have similar size and shape as Gln. As a control, Asn was introduced at Gln41 as well. The effects of these amino acid substitutions on the stability, and in particular, on the heat capacity change upon unfolding were measured using differential scanning calorimetry. The effect of the amino acid substitutions on the structure was also evaluated by comparing the (1)H-(15)N HSQC spectra of the ubiquitin variants. It was found that the Ala substitutions did not have a considerable effect on the heat capacity change upon unfolding. However, the substitutions of aliphatic side chains (Val or Leu) with a polar residue (Asn) lead to a significant (> 30%) decrease in the heat capacity change upon unfolding. The decrease in heat capacity changes does not appear to be the result of significant structural perturbations as seen from the HSQC spectra of the variants. The substitution of a buried polar residue (Gln41) to a nonpolar residue (Leu or Val) leads to a significant (> 25%) increase in heat capacity change upon unfolding. These results indicate that indeed the heat capacity change of burial of polar and nonpolar groups has an opposite sign. However, the observed changes in DeltaC(p) are several times larger than those predicted, based on the changes in water accessible surface area upon substitution.     

Thermodynamic Characterization of the Unfolding of the Prion Protein

The prion protein appears to be unusually susceptible to conformational change, and unlike nearly all other proteins, it can easily be made to convert to alternative misfolded conformations. To understand the basis of this structural plasticity, a detailed thermodynamic characterization of two variants of the mouse prion protein (moPrP), the full-length moPrP (23–231) and the structured C-terminal domain, moPrP (121–231), has been carried out. All thermodynamic parameters governing unfolding, including the changes in enthalpy, entropy, free energy, and heat capacity, were found to be identical for the two protein variants. The N-terminal domain remains unstructured and does not interact with the C-terminal domain in the full-length protein at pH 4. Moreover, the enthalpy and entropy of unfolding of moPrP (121–231) are similar in magnitude to values reported for other proteins of similar size. However, the protein has an unusually high native-state heat capacity, and consequently, the change in heat capacity upon unfolding is much lower than that expected for a protein of similar size. It appears, therefore, that the native state of the prion protein undergoes substantial fluctuations in enthalpy and hence, in structure.     

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.     

Heat capacity changes associated with DNA duplex formation: salt- and sequence-dependent effects

Duplexes are the most fundamental elements of nucleic acid folding. Although it has become increasingly clear that duplex formation can be associated with a significant change in heat capacity (deltaC(p)), this parameter is typically overlooked in thermodynamic studies of nucleic acid folding. Analogy to protein folding suggests that base stacking events coupled to duplex formation should give rise to a deltaC(p) due to the release of waters solvating aromatic surfaces of nucleotide bases. In previous work, we showed that the deltaC(p) observed by isothermal titration calorimetry (ITC) for RNA duplex formation depended on salt and sequence [Takach, J. C., Mikulecky, P. J., and Feig, A. L. (2004) J. Am. Chem. Soc. 126, 6530-6531]. In the present work, we apply calorimetric and spectroscopic techniques to a series of designed DNA duplexes to demonstrate that both the salt dependence and sequence dependence of deltaC(p)s observed by ITC reflect perturbations to the same fundamental phenomenon: stacking in the single-stranded state. By measuring the thermodynamics of single strand melting, one can accurately predict the deltaC(p)s observed for duplex formation by ITC at high and low ionic strength. We discuss our results in light of the larger issue of contributions to deltaC(p) from coupled equilibria and conclude that observed deltaC(p)s can be useful indicators of intermediate states in nucleic acid folding phenomena.     

Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes

Directed evolution techniques have been used to improve the thermal stability of the xylanase A from Bacillus subtilis (XylA). Two generations of random mutant libraries generated by error prone PCR coupled with a single generation of DNA shuffling produced a series of mutant proteins with increasing thermostability. The most Thermostable XylA variant from the third generation contained four mutations Q7H, G13R, S22P, and S179C that showed an increase in melting temperature of 20 degrees C. The thermodynamic properties of a representative subset of nine XylA variants showing a range of thermostabilities were measured by thermal denaturation as monitored by the change in the far ultraviolet circular dichroism signal. Analysis of the data from these thermostable variants demonstrated a correlation between the decrease in the heat capacity change (deltaC(p)) with an increase in the midpoint of the transition temperature (T(m)) on transition from the native to the unfolded state. This result could not be interpreted within the context of the changes in accessible surface area of the protein on transition from the native to unfolded states. Since all the mutations are located at the surface of the protein, these results suggest that an explanation of the decrease in deltaC(p) should include effects arising from the protein/solvent interface.     

Thermodynamics of the Trp‐cage miniprotein unfolding in urea

The thermodynamic properties of unfolding of the Trp‐cage mini protein in the presence of various concentrations of urea have been characterized using temperature‐induced unfolding monitored by far‐UV circular dichroism spectroscopy. Analysis of the data using a two‐state model allowed the calculation of the Gibbs energy of unfolding at 25°C as a function of urea concentration. This in turn was analyzed by the linear extrapolation model that yielded the dependence of Gibbs energy on urea concentration, i.e. the m‐value for Trp‐cage unfolding. The m‐value obtained from the experimental data, as well as the experimental heat capacity change upon unfolding, were correlated with the structural parameters derived from the three dimensional structure of Trp‐cage. It is shown that the m‐value can be predicted well using a transfer model, while the heat capacity changes are in very good agreement with the empirical models based on model compounds studies. These results provide direct evidence that Trp‐cage, despite its small size, is an excellent model for studies of protein unfolding and provide thermodynamic data that can be used to compare with atomistic computer simulations. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Stabilization of proteins by enhancement of inter-residue hydrophobic contacts: lessons of T4 lysozyme and barnase

Although the hydrophobic interactions are considered as the main contributors to the protein stability, not much examples of protein stabilization by rational increasing of this type of interactions still can be found in literature. This is partly due to the lack of proper theoretical "measure" of hydrophobic interactions and their changes upon mutations. In the present paper the molecular hydrophobicity potential approach is used to assess how the changes in type and the strength of inter-residue contacts upon single amino acid mutations are correlated with the changes in thermodynamic stability of T4 lysozyme and barnase mutants, and which factors affect these correlations. Mutations changing unfavorable hydrophilic-to-hydrophobic contacts into favorable hydrophobic were found to enhance the thermodynamic stability in more than 81 % of cases, if these mutations do not create steric bumps and do not involve proline residues and hydrogen-bonded side-chains. Mutations increasing hydrophobic contributions (according to molecular hydrophobicity potential formalism) lead to increase of thermodynamic stability in more than 94% of cases for certain type of mutations (i.e., mutations not involving charged residues, Pro and residues with side-chain hydrogen bonds, when these mutations do not introduce steric bumps and do not involve strongly exposed residues and residues situated at helix N- and C-cap positions). For this type of mutations the correlation was found between the change in hydrophobic contributions of mutated residues deltaCphob and thermodynamic parameters deltaTm (change in melting temperature) and deltadeltaG (change in free energy of unfolding). Although the correlation coefficients were larger if the experimental structures of mutants were used for the calculations (correlation coefficients r(exp) deltaC,deltaT = .85 and r(exp) deltaC,deltadeltaG = 0.87) than if the modeled structures were used instead (r(mod) deltaC,deltaT = 0.74 and r(mod)deltaC,deltadeltaG = 0.76), the modelled structures of mutants in the vast majority of cases can be used for qualitative predictition of the protein stabilization. Basing on the analysis of mutations increasing hydrophobic contributions in T4 lysozyme the substitution matrix was derived, which can be used to decide which new residue should be put instead the old one to increase the stability of protein. The estimation shows that the number of potential mutation sites for enhancement of hydrophobic interactions in T4 lysozyme is quite large, and only approximately 10 per cent of them were studied thus far. Basing on the current analysis of T4 lysozyme and barnase mutations the algorithm for increasing of protein stability via increasing of hydrophobic interactions for the proteins with known spatial structure is proposed.     

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.     

Stabilization of alpha-chymotrypsin upon PEGylation correlates with reduced structural dynamics

Protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. Poly(ethylene glycol) (PEG) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. The observed increase in thermodynamic stability of several proteins upon PEGylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing. To test this hypothesis, the model protein alpha-chymotrypsin (alpha-CT) was covalently modified with PEGs with molecular weights (M(W)) of 700, 2,000 and 5,000 and the degree of modification was systematically varied. The procedure did not cause significant tertiary structure changes. Thermodynamic unfolding experiments revealed that PEGylation increased the thermal transition temperature (T(m)) of alpha-CT by up to 6 degrees C and the free energy of unfolding [DeltaG(U) (25 degrees C)] by up to 5 kcal/mol. The increase in stability was found to be independent of the PEG M(W) and it leveled off after an average of four PEG molecules were bound to alpha-CT. Fourier-transformed infrared (FTIR) H/D exchange experiments were conducted to characterize the conformational dynamics of the PEG-conjugates. It was found that the magnitude of thermodynamic stabilization correlates with a reduction in protein structural dynamics and was independent of the PEG M(W). Thus, the initial hypothesis proved positive. Similar to the thermodynamic stabilization of proteins by covalent modification with glycans, PEG thermodynamically stabilizes alpha-CT by reducing protein structural dynamics. These results provide guidance for the future development of stable protein formulations.     

Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding.

Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (delta Cp), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and delta Cp.     

Solvent isotope effect on thermodynamics of hydration

Partial molar heat capacities of five linear alcohols (methanol, ethanol, n-propanol, n-butanol, n-pentanol) and five N-substituted amides (n-propionamide, N-methylformamide, N-methylacetamide, N-methylpropionamide, N-ethylacetamide) in aqueous D(2)O solution have been measured at 25 degrees C. The heat capacities of transfer of these compounds from H(2)O to D(2)O were calculated using previously reported (Makhatadze et al., Biophys. Chem. 64 (1997) 93) values of partial heat capacities of alcohols and amides in aqueous H(2)O solutions. It is shown that the sign and magnitude of the heat capacity change upon transfer from H(2)O to D(2)O depends on the relative amount of polar and non-polar solvent accessible surface areas of solute. Analysis shows that transfer of non-polar surface from H(2)O to D(2)O is accompanied by a positive heat capacity change. In contrast, transfer of polar surface from H(2)O to D(2)O occurs with negative heat capacity change. Estimates show that the solvent isotope effect on the heat capacity changes upon protein unfolding can be predicted using the changes of the polar and non-polar surface area changes upon protein unfolding and the transfer data of model compounds. Analysis of the thermodynamic functions of transfer of non-polar compounds from H(2)O to D(2)O shows puzzling behavior which contradicts current definitions of the hydrophobic effect.     

Electrostatic interactions contribute to reduced heat capacity change of unfolding in a thermophilic ribosomal protein l30e

The origin of reduced heat capacity change of unfolding (DeltaC(p)) commonly observed in thermophilic proteins is controversial. The established theory that DeltaC(p) is correlated with change of solvent-accessible surface area cannot account for the large differences in DeltaC(p) observed for thermophilic and mesophilic homologous proteins, which are very similar in structures. We have determined the protein stability curves, which describe the temperature dependency of the free energy change of unfolding, for a thermophilic ribosomal protein L30e from Thermococcus celer, and its mesophilic homologue from yeast. Values of DeltaC(p), obtained by fitting the free energy change of unfolding to the Gibbs-Helmholtz equation, were 5.3 kJ mol(-1) K(-1) and 10.5 kJ mol(-1) K(-1) for T.celer and yeast L30e, respectively. We have created six charge-to-neutral mutants of T.celer L30e. Removal of charges at Glu6, Lys9, and Arg92 decreased the melting temperatures of T.celer L30e by approximately 3-9 degrees C, and the differences in melting temperatures were smaller with increasing concentration of salt. These results suggest that these mutations destabilize T.celer L30e by disrupting favorable electrostatic interactions. To determine whether electrostatic interactions contribute to the reduced DeltaC(p) of the thermophilic protein, we have determined DeltaC(p) for wild-type and mutant T.celer L30e by Gibbs-Helmholtz and by van't Hoff analyses. A concomitant increase in DeltaC(p) was observed for those charge-to-neutral mutants that destabilize T.celer L30e by removing favorable electrostatic interactions. The crystal structures of K9A, E90A, and R92A, were determined, and no structural change was observed. Taken together, our results support the conclusion that electrostatic interactions contribute to the reduced DeltaC(p) of T.celer L30e.     

Elimination of the C-cap in ubiquitin - structure, dynamics and thermodynamic consequences

Single amino acid substitutions rarely produce substantial changes in protein structure. Here we show that substitution of the C-cap residue in the alpha-helix of ubiquitin with proline (34P variant) leads to dramatic structural changes. The resulting conformational perturbation extends over the last two turns of the alpha-helix and leads to enhanced flexibility for residues 27-37. Thermodynamic analysis of this ubiquitin variant using differential scanning calorimetry reveals that the thermal unfolding transition remains highly cooperative, exhibiting two-state behavior. Similarities with the wild type in the thermodynamic parameters (heat capacity change upon unfolding and m-value) of unfolding monitored by DSC and chemical denaturation suggests that the 34P variant has comparable buried surface area. The hydrophobic core of 34P variant is not packed as well as that of the wild type protein as manifested by a lower enthalpy of unfolding. The increased mobility of the polypeptide chain of this ubiquitin variant allows the transient opening of the hydrophobic core as evidenced by ANS binding. Taken together, these results suggest exceptional robustness of cooperativity in protein structures.     

A study on the enthalpy-entropy compensation in protein unfolding

A large number of thermodynamic data including the free energy, enthalpy, entropy, and heat capacity changes were collected for the denaturation of various proteins. Regression indicated that remarkable enthalpy-entropy compensation occurred in protein unfolding, which meant that the change in enthalpy was almost compensated by a corresponding change in entropy resulting in a smaller net free energy change. This behavior was proposed to result from the water molecule reorganization, which contributed significantly to the enthalpy and entropy changes but little to the free energy change in protein unfolding. It turned out that the enthalpy-entropy compensation could provide novel insights into the problem of enthalpy and entropy convergence in protein unfolding.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

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.     

Thermal denaturation of iso-1-cytochrome c variants: comparison with solvent denaturation.

Thermal denaturation studies as a function of pH were carried out on wild-type iso-1-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence. By examining the enthalpy and Tm at various pH values, the heat capacity increment (delta Cp), which is dominated by the degree of change in nonpolar hydration upon protein unfolding, was found for the wild type where lysine 73 is normally present and for three variants. For the Trp 73 variant, the delta Cp value (1.15 +/- 0.17 kcal/mol K) decreased slightly relative to wild-type iso-1-cytochrome c (1.40 +/- 0.06 kcal/mol K), while for the Ile 73 (1.65 +/- 0.07 kcal/mol K) and the Val 73 (1.50 +/- 0.06 kcal/mol K) variants, delta Cp increased slightly. In previous studies, the Trp 73, Ile 73, and Val 73 variants have been shown to have decreased m-values in guanidine hydrochloride denaturations relative to the wild-type protein (Hermann L, Bowler BE, Dong A, Caughey WS. 1995. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: Investigation of aliphatic residues. Biochemistry 34:3040-3047). Both the m-value and delta Cp are related to the change in solvent exposure upon unfolding and other investigators have shown a correlation exists between these two parameters. However, for this subset of variants of iso-1-cytochrome c, a lack of correlation exists which implies that there may be basic differences between the guanidine hydrochloride and thermal denaturations of this protein. Spectroscopic data are consistent with different denatured states for thermal and guanidine hydrochloride unfolding. The different response of m-values and delta Cp for these variants will be discussed in this context.     

Unfolding thermodynamics of Trp-cage, a 20 residue miniprotein, studied by differential scanning calorimetry and circular dichroism spectroscopy

Small proteins provide convenient models for computational studies of protein folding and stability, which are usually compared with experimental data. Until recently, the unfolding of Trp-cage was considered to be a two-state process. However, no direct experimental evidence for this has been presented, and in some cases, the contrary has been suggested. To elucidate a detailed unfolding mechanism, we studied the thermodynamics of unfolding of Trp-cage by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. The observation that at low temperatures only approximately 90-95% of Trp-cage exists in the native conformation presented an analytical challenge. Nevertheless, it was found that the DSC and CD data can be fitted simultaneously to the same set of thermodynamic parameters. The major uncertainty in such a global fit is the heat capacity change upon unfolding, DeltaCp. This can be circumvented by obtaining DeltaCp directly from the difference between heat capacity functions of the native and unfolded states. Using such an analysis it is shown that Trp-cage unfolding can be represented by a two-state model with the following thermodynamic parameters: Tm = 43.9 +/- 0.8 degrees C, DeltaH(Tm) = 56 +/- 2 kJ/mol, DeltaCp = 0.3 +/- 0.1 kJ/(mol.K). Using these thermodynamic parameters it is estimated that Trp-cage is marginally stable at 25 degrees C, DeltaG(25 degrees C) = 3.2 +/- 0.2 kJ/mol, which is only 30% more than the thermal fluctuation energy at this temperature.