Thermodynamic analysis of denaturant-induced unfolding of HodC69S protein supports a three-state mechanism.

Thermodynamic stability parameters and the equilibrium unfolding mechanism of His 6HodC69S, a mutant of 1 H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) having a Cys to Ser exchange at position 69 and an N-terminal hexahistidine tag (His 6HodC69S), have been derived from isothermal unfolding studies using guanidine hydrochloride (GdnHCl) or urea as denaturants. The conformational changes were monitored by following changes in circular dichroism (CD), fluorescence, and dynamic light scattering (DLS), and the resulting transition curves were analyzed on the basis of a sequential three-state model N = I = D. The structural changes have been correlated to catalytic activity, and the contribution to stability of the disulfide bond between residues C37 and C184 in the native protein has been established. A prominent result of the present study is the finding that, independent of the method used for denaturing the protein, the unfolding mechanism always comprises three states which can be characterized by, within error limits, identical sets of thermodynamic parameters. Apparent deviations from three-state unfolding can be rationalized by the inability of a spectroscopic probe to discriminate clearly between native, intermediate, and unfolded ensembles. This was the case for the CD-monitored urea unfolding curve.

Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2.

The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5 degrees C and 59.9 degrees C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transitions of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies DeltaG(0)(D)(buffer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, K(d), for wild-type SCP2 are 335 microM at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpies, DeltaH(b), are -21. 5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degrees C. The parameters for the c71s mutant at 25 degrees C are K(d)=413 microM and DeltaH(b)=16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity.

Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters.

The stability parameters delta Gst, delta Hst and delta Sst of native basic pancreatic trypsin inhibitor (BPTI) have been characterized by microcalorimetric unfolding studies in various buffer solutions, at different pH values and in the presence of guanidine hydrochloride. The unfolding enthalpy of BPTI, in contrast ot other globular proteins, exhibits a very small dependence on temperature, which results in a characteristic different temperature dependence of the Gibbs energy of stabilization. BPTI has a very high specific Gibbs energy of stabilization, which renders the slow exchange rates of amide protons understandable. Comparison of the unfolding entropy of BPTI at 110 degrees C with corresponding values of other proteins, revealed that the delta S values of BPTI are lower by 2.9 J/(K X residue). This lower value of the unfolding entropy is in good agreement with predictions of a theoretical study by Poland & Scheraga (1965) where the influence of crosslinks on the configurational entropy has been studied. Additionally, we were able to calculate an interaction enthalpy per site of -5.6 kJ/mol based on the measurements of unfolding of BPTI in 6 M-guanidine hydrochloride.

Folding energetics of ligand binding proteins. I. Theoretical model.

Heat capacity curves as obtained from differential scanning calorimetry are an outstanding source for molecular information on protein folding and ligand-binding energetics. However, deconvolution of C(p) data of proteins in the presence of ligands can be compromised by indeterminacies concerning the correct choice of the statistical thermodynamic ensemble. By convent, the assumption of constant free ligand concentration has been used to derive formulae for the enthalpy. Unless the ligand occurs at large excess, this assumption is incorrect. Still the relevant ensemble is the grand canonical ensemble. We derive formulae for both constraints, constancy of total or free ligand concentration and illustrate the equations by application to the typical equilibrium Nx <=> N + x <=> D + x. It is demonstrated that as long as the thermodynamic properties of the ligand can be completely corrected for by performing a reference measurement, the grand canonical approach provides the proper and mathematically significantly simpler choice. We demonstrate on the two cases of sequential or independent ligand-binding the fact, that similar binding mechanisms result in different and distinguishable heat capacity equations. Finally, we propose adequate strategies for DSC experiments as well as for obtaining first estimates of the characteristic thermodynamic parameters, which can be used as starting values in a global fit of DSC data.

A new set of peptide-based group heat capacities for use in protein stability calculations.

The partial molar heat capacities of the tripeptides of the sequence glycyl-X-glycine, where X is one of the amino acids leucine, threonine, glutamine, phenylalanine, histidine, cysteine, proline, glutamic acid or arginine, and of the two tetrapeptides tetraglycine and glycyltryptophanylglycylglycine in aqueous solution over the temperature range 10-100 degrees C have been determined using high sensitivity scanning microcalorimetry. These results were used to derive the partial molar heat capacities of the various amino acid side-chains. This report completes our programme to derive reliable side-chain heat capacities for all 20 amino acids of proteins over a wide temperature range using the tripeptides Gly-X-Gly as realistic model compounds. Included in the study is a summary of the partial molar heat capacities of all 20 amino acid side-chains. These results, along with the heat capacity of the peptide backbone group, were used to calculate the partial molar heat capacities of some oligopeptides and of the random coil form of some unfolded proteins in water. The calculated heat capacities of the proteins obtained using this new set of heat capacities for the constituent groups are consistent with the heat capacities of the denatured state determined experimentally.

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.

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.

Folding energetics of ligand binding proteins II. Cooperative binding of Ca2+ to annexin I.

The calcium binding properties of annexin I as observed by thermodynamic DSC studies have been compared to the structural information obtained from X-ray investigation. The calorimetric experiment permitted to evaluate both the reaction scheme - including binding of ligand and conformational changes - and the energetics of each reaction step. According to published X-ray data Annexin I has six calcium binding sites, three medium-affinity type II and three low-affinity type III sites. The present study shows that at 37 degrees C annexin I binds in a Hill type fashion simultaneously two calcium ions in a first step with medium affinity at a concentration of 0.6 mM and another three Ca(2+) ions again cooperatively at 30 mM with low affinity. Therefore it can be concluded that only two medium-affinity type II binding sites are available. The third site, that should be accessible in principle appears to be masked presumably due to the presence of the N terminus. In view of the large calcium concentration needed for saturation of the binding sites, annexin I may be expected to be Ca(2+) free in vivo unless other processes such as membrane interaction occur simultaneously. This assumption is consistent with the finding, that the affinity of annexins to calcium is usually markedly increased by the presence of lipids.

Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the alpha-amylase inhibitor tendamistat.

The present differential scanning calorimetry and circular dichroism studies on the mechanism of protein stabilization by disulfide bonds were concerned with two questions: is the increase in unfolding entropy upon removal of disulfide links sufficient for the explantation of the general stability decrease of disulfide-deficient mutants? Is it immaterial by which residue cysteine residues are replaced when disulfide bridges are to be opened? To answer these questions we investigated two disulfide bridge mutants of the alpha-amylase inhibitor Tendamistat where the large loop (C45A/C73A) or the small loop (C11A/C27A) had been opened by recombinant DNA techniques, and we compared the stability of the mutated proteins with that of wild-type Tendamistat published previously. To elucidate the significance of the nature of the group that replaces Cys we introduced in position 27 of the small loop four different amino acids instead of Cys: Ala, Leu, Ser and Thr. Surprisingly, opening of the small loop (17 residues) causes larger destabilization than opening of the large loop comprising 29 residues. The thermodynamic parameters at pH 7.0 are: wild-type: t1/2 = 81.6 degrees C, delta Hcal = 296 kJ mol-1, large loop mutant (C45A/C73A): t1/2 = 58.6 degrees C, delta Hcal = 225 kJ mol-1 and small loop mutant (C11A/C27A): t1/2 = 42.7 degrees C, delta Hcal = 135 kJ mol-1. This finding is at variance with the entropy hypothesis. The relative contributions to stability of enthalpic and entropic terms can be varied by a proper choice of substitutions. While the destabilization originating from C45A/C73A exchanges in the large loop turns out to be purely entropic, the stability decreases of the small loop mutants are caused by changes in both enthalpic and entropic terms. Leu or Ser in position 27 leads to an overall enthalpic destabilization. Thr in position 27 increases the transition enthalpy of this mutant to the value of the wild-type protein but increases at the same time the value of the transition entropy with the result of an overall entropic destabilization. Finally, in the C11A/C27A small loop mutant of lowest stability a very large enthalpic destabilization occurs, which is, however, partly counterbalanced by a reduction in the transition entropy. The preferential perturbation of the native state by the mutations is manifest in the increase of the native state heat capacity relative to that of the wild-type protein and the identity of the heat capacity of the unfolded state.(ABSTRACT TRUNCATED AT 400 WORDS)

Dimer-to-tetramer transformation: loop excision dramatically alters structure and stability of the ROP four alpha-helix bundle protein.

The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-alpha-helix motif of the wild-type structure is given up. Instead a homotetrameric four-alpha-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71 degreesC characteristic of the wild-type protein to 101 degreesC for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer)-1 to 195.1 kJ (mol of tetramer)-1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of DeltaH=1073+/-30 kJ (mol of tetramer)-1 and a heat capacity change at the transition temperature of DeltaDNCp=14.9(+/-)3% kJ (mol of tetramerxK)-1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van't Hoff enthalpy values.

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.

Extreme thermostability of tarantula hemocyanin.

Biotops with extreme temperatures such as deserts force animals to avoid or escape high temperatures by biochemical, behavioural or morphological adaptation. In this context we tested the resistance to heat of the oxygen carrier hemocyanin from the ancient tarantula Eurypelma californicum, which is found in arid zones of North America. Differential scanning calorimetry, light scattering, crossed immunogelelectrophoresis and oxygen binding experiments show that the 24-meric hemocyanin is conformationally stable and fully functioning at temperatures up to 90 degrees C. Our results demonstrate that the cation-mediated state of oligomerization is not only crucial for the high cooperativity of oxygen binding of this hemocyanin, but also for its extreme stability in the physiological temperature and pH range.

Ca2+-independent interaction of annexin I with phospholipid monolayers.

At pH 6.0, the interaction of annexin I, a proteolytic fragment of annexin I and annexin V, was studied with monolayers composed of dipalmitoylphosphatidylserine (DPPS), dipalmitoylphosphatidylcholine (DPPC) or DPPS/DPPC mixtures (molar ratio 1:4). The measurements reveal that only annexin I shows a significant increase in the surface pressure at constant surface area in the absence of Ca2+ ions. We interpret these pressure changes as reflecting penetration of the protein. Kinetic analyses of the annexin I/monolayer interaction at pH 6.0 in the presence and absence of Ca2+ ions show differences between the interaction mechanisms that support the occurrence of a pH-regulated process. At pH 7.4, Ca2+ ions are required for the interaction.

Acid induced equilibrium unfolding of annexin V wild type shows two intermediate states.

Annexin V is an alpha-helical protein which shows anticoagulatory and antiinflammatory activity. It is supposed to be involved in membrane fusion and exocytosis. In this study acid-induced equilibrium unfolding of the human annexin V is investigated by fluorescence and circular dichroism spectroscopy. The spectroscopic data indicate that at least two intermediate states are involved in unfolding. One of the proposed intermediate states exhibits properties similar to those observed with annexin V wild type saturated with calcium, another may be regarded as 'molten globule'.

Response functions of proteins.

The thermodynamics of protein folding can be studied by a variety of different techniques such as differential scanning calorimetry, differential scanning densimetry and sound velocity measurements. These three methods monitor the different response functions heat capacity, expansion coefficient and compressibility that characterise various aspects of protein dynamics such as equilibrium energy and volume fluctuations and energy-volume correlations. For the development of a comprehensive thermodynamic description of protein behaviour information on these response functions should be combined. As a starting point we provide in the present paper analytical solutions for the determination of the response functions and demonstrate on several examples how to extract a maximum of thermodynamic information on proteins from the measurements of Cp, alpha p and kappa T.

Protein heat capacity: inconsistencies in the current view of cold denaturation.

The present study shows on the basis of the thermodynamic stability criterion (partial differential S/partial differential T)p>0 that partitioning of the entropy of cold-unfolding of a protein into independent positive conformational and negative hydrational contributions is incorrect. Furthermore it provides a new microscopic interpretation of protein heat capacity that takes into account the significant fluctuations in energy and entropy which result from the small size of these macromolecules.

Partial molar heat capacities and volumes of Gly-X-Gly tripeptides in aqueous solution: model studies for the rationalization of thermodynamic parameters of proteins.

The thermodynamics of protein unfolding can be rationalized if the temperature dependence of the partial molar volumes and heat capacities of their constituent groups are known reliably. Despite many experimental and theoretical studies there are still several inconsistencies in the published thermodynamic data. We have investigated some of these inconsistencies by applying high sensitivity scanning densimetry and microcalorimetry to aqueous solutions of tripeptides of the structure Gly-X-Gly, where X is one of the amino acids Met, Asn, Gly and Ile. For these side-chains either no direct data have been determined or serious discrepancies exist between the values published by different laboratories. Partial molar heat capacities and volumes have been determined for the peptides in pure water, in water adjusted to pH = 4 and in 0.5 M sodium acetate buffer at pH = 4. The results obtained are critically compared with those in the literature.

The 'Janus' nature of proteins: systems at the verge of the microscopic and macroscopic world.

Direct measurement of the heat capacity of proteins by microcalorimetry has had a decisive impact on understanding the behaviour of these biopolymers. Statistical mechanics allow a straightforward calculation and prediction of the enthalpy and heat capacity curves from the partition function. We show that these predictions can differ from the more intuitive models used so far for the description of the thermodynamic behaviour of proteins if the transition involves a stoichiometry other than 1:1. Furthermore, we delineate that the characteristics of protein unfolding are governed by the fluctuations associated with the small size of these molecules. Therefore it may be necessary to modify the picture of the unfolding of small proteins in the light of statistical physics, while for very large proteins the current view may be maintained as a useful limiting approximation.

An alternative interpretation of the heat capacity changes associated with protein unfolding.

The present study devises a method to get quantitative information for proteins on the theoretically important heat capacity at constant volume. For this purpose expansion coefficients of both the native and unfolded state of a variety of proteins have been determined and used together with compressibility coefficients to calculate the difference between isobaric and isochoric heat capacity, (cp - cv), for the unfolding transition. This difference delta (cp - cv) = (c - c) - (c - c) turns out to be a positive number that is larger than the experimental isobaric heat capacity change (formula:see text) [corrected] for the proteins studied. The generally observed positive heat capacity change on unfolding can therefore alternatively be interpreted as resulting from the difference in work involved in changing the intra- and intermolecular interactions including the weak, highly distance-dependent, van der Waals interactions, for the unfolded and native state, respectively. The difference in expansion work against the atmospheric pressure is negligible. This macroscopic interpretation cannot rule out that part of the denaturational heat capacity increase is also due to the different interaction with water of the native and unfolded conformations of the protein.

Partial molar volumes of proteins: amino acid side-chain contributions derived from the partial molar volumes of some tripeptides over the temperature range 10-90 degrees C.

The partial molar volumes of tripeptides of sequence glycyl-X-glycine, where X is one of the amino acids alanine, leucine, threonine, glutamine, phenylalanine, histidine, cysteine, proline, glutamic acid, and arginine, have been determined in aqueous solution over the temperature range 10-90 degrees C using differential scanning densitometry . These data, together with those reported previously, have been used to derive the partial molar volumes of the side-chains of all 20 amino acids. The side-chain volumes are critically compared with literature values derived using partial molar volumes for alternative model compounds. The new amino acid side-chain volumes, along with that for the backbone glycyl group, were used to calculate the partial specific volumes of several proteins in aqueous solution. The results obtained are compared with those observed experimentally. The new side-chain volumes have also been used to re-determine residue volume changes upon protein folding.