Thermal unfolding of plastocyanin from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 and comparison with its thermophilic counterpart from Phormidium laminosum

The thermal unfolding of plastocyanin from the mesophilic cyanobacterium Synechocystis is described herein, and the results are compared with those obtained for the homologous thermophilic protein from Phormidium laminosum. The thermal unfolding is irreversible under all the conditions that were investigated. Plastocyanin from the thermophilic organism, both in the native state and in the apoprotein form, proved to be more thermostable than its mesophilic counterpart under all experimental conditions. Synechocystis reduced plastocyanin has been shown to be more stable than the oxidized species, both with respect to the required temperature for protein unfolding and with respect to the kinetics of the process. This behavior contrasts with that observed for Phormidium plastocyanin, in which the oxidized form is the more stable one. The unfolding pH dependence and kinetic studies indicate that around physiological pH, the most kinetically stable form is also the one more resistant to temperature variations, suggesting a close compromise between function and stability. Molecular dynamics simulations suggest that Phormidium and Synechocystis plastocyanins follow different unfolding pathways that affect different protein areas and which could be responsible for the observed dissimilar thermal resistance.     

Thermodynamics elucidation of the structural stability of a thermophilic protein

The structural stability of the protein, phycocyanin isolated from two strains of cyanophyta, Synechococcus lividus (thermophile) and Phormidium luridum (mesophile), are investigated by comparative thermal and denaturant unfolding, using differential scanning calorimetry, visible absorption spectrophotometry, and circular dichroism. The thermophilic protein exhibits a much higher temperature and enthalpy of unfolding from the native to the denatured state. The concentration of urea at half-completion of thermal unfolding is essentially the same between the thermophilic and mesophilic proteins; in contrast, the corresponding temperature and the enthalpy of thermal unfolding are much higher for the thermophilic protein. In addition, the concentration of urea at which the non-thermal (denaturant) unfolding of protein is half-completed, as detected by either circular dichroism or absorption spectroscopy, is significantly higher in the thermophilic protein, while the apparent free energy of unfolding only shows a moderate difference between the two proteins. The distinct differences in the enthalpy of thermal unfolding and the free energy of denaturant unfolding are interpreted in terms of a significant entropy change associated with the unfolding of these proteins. This entropy contribution is much higher in the thermophilic protein, and may be derived from its more rigid overall structure that possesses higher internal hydrophobicity and stronger internal packing.     

In vivo photosystem I reduction in thermophilic and mesophilic cyanobacteria: the thermal resistance of the process is limited by factors other than the unfolding of the partners

Photosystem I reduction by plastocyanin and cytochrome c(6) in cyanobacteria has been extensively studied in vitro, but much less information is provided on this process inside the cell. Here, we report an analysis of the electron transfer from both plastocyanin and cytochrome c(6) to photosystem I in intact cells of several cyanobacterial species, including a comparative study of the temperature effect in mesophilic and thermophilic organisms. Our data show that cytochrome c(6) reduces photosystem I by following a reaction mechanism involving complex formation, whereas the copper-protein follows a simpler collisional mechanism. These results contrast with previous kinetic studies in vitro. The effect of temperature on photosystem I reduction leads us to conclude that the thermal resistance of this process is determined by factors other than the proper stability of the protein partners.     

Thermal stability of wild type and disulfide bridge containing mutant of poplar plastocyanin

A comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning calorimetry and optical spectroscopic techniques. For wild type plastocyanin the transition temperature, determined from the calorimetric profiles, is 62.7 degrees C at the scan rate of 60 degrees C/h, whereas for the mutant it is reduced to 58.0 degrees C. In both cases, the endothermic peak is followed by an exothermic one at higher temperatures. The unfolding process monitored by optical absorption at 596 nm also reveals a reduced thermal stability of the mutated plastocyanin compared to the wild type protein, with transition temperatures of 54.8 and 58.0 degrees C, respectively. For both proteins, the denaturation process was found to be irreversible and dependent on the scan rate preventing the thermodynamic analysis of the unfolding process. In parallel, small conformational changes between wild type and mutant plastocyanin emerge from fluorescence spectroscopy measurements. Here, a difference in the interaction of the two proteins between the microenvironment surrounding the fluorophores and the solvent was proposed. The destabilization observed in the disulfide containing mutant of plastocyanin suggests that the double mutation, Ile21Cys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink.     

Probing the mechanism of rubredoxin thermal unfolding in the absence of salt bridges by temperature jump experiments

Rubredoxins are the simplest type of iron-sulphur proteins and in recent years they have been used as model systems in protein folding and stability studies, especially the proteins from thermophilic sources. Here, we report our studies on the rubredoxin from the hyperthermophile Methanococcus jannaschii (T opt = 85 degrees C), which was investigated in respect to its thermal unfolding kinetics by temperature jump experiments. Different spectroscopic probes were used to monitor distinct structural protein features during the thermal transition: the integrity of the iron-sulphur centre was monitored by visible absorption spectroscopy, whereas tertiary structure was followed by intrinsic tryptophan fluorescence and exposure of protein hydrophobic patches was sensed by 1-anilinonaphthalene-8-sulphonate fluorescence. The studies were performed at acidic pH conditions in which any stabilising contributions from salt bridges are annulled due to protonation of protein side chain groups. In these conditions, M. jannaschii rubredoxin assumes a native-like, albeit more flexible and open conformation, as indicated by a red shift in the tryptophan emission maximum and 1-anilinonaphthalene-8-sulphonate binding. Temperature jumps were monitored by the three distinct techniques and showed that the protein undergoes thermal denaturation via a simple two step mechanism, as loss of tertiary structure, hydrophobic collapse, and disintegration of the iron-sulphur centre are concomitant processes. The proposed mechanism is framed with the multiphasic one proposed for Pyrococcus furiosus rubredoxin, showing that a common thermal unfolding mechanism is not observed between these two closely related thermophilic rubredoxins.     

Tracking the unfolding and refolding pathways of outer membrane protein porin from Paracoccus denitrificans

We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin.     

Exploiting the Link between Protein Rigidity and Thermostability for Data‐Driven Protein Engineering

Understanding and exploiting the relationship between microscopic structure and macroscopic stability is important for developing strategies to improve protein stability at high temperatures. The thermostability of proteins has been repeatedly linked to an enhanced structural rigidity of the folded native state. In the current study, the rigidity of protein structures from mesophilic and thermophilic organisms along a thermal unfolding trajectory is directly probed. In order to perform this, protein structures were modeled as constraint networks, and the rigidity in these networks was quantified using the Floppy Inclusion and Rigid Substructure Topography (FIRST) method. During the thermal unfolding, a phase transition was observed that defines the rigidity percolation threshold and corresponds to the folded‐unfolded transition in protein folding. Using concepts from percolation theory and network science, a higher phase transition temperature was observed for ca. two‐thirds of the proteins from thermophilic organisms compared to their mesophilic counterparts, when applied to a data set of 20 pairs of homologues. From both the analysis of the microstructure of the constraint networks and monitoring the macroscopic behavior during the thermal unfolding, direct evidence was found for the “corresponding states” concept, which states that mesophilic and thermophilic enzymes are in corresponding states of similar flexibility at their respective optimal temperature. Finally, the current approach facilitated the identification of structural features from which a destabilization of the structure originates upon thermal unfolding. These predictions show a good agreement with the experimental data. Therefore, the information might be exploited in data‐driven protein engineering by pointing to residues that should be varied to obtain a protein with higher thermostability.     

Stability and folding of the ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric protein containing two iron-sulfur centres, one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+). It is an intrinsically hyperstable protein, being expressed at the organism's extreme optimal growth temperature: 80 degrees C. Using spectroscopic methods we have investigated the unfolding reaction of the Acidianus ambivalens ferredoxin. No unfolding of the oxidised ferredoxin was observed at pH 7.0, even in the presence of 8 M GuHCl. Upon increasing the pH to 10.0, the unfolding transition showed a midpoint at 6.3 M GuHCl and an unfolding-free energy of 70 kJ mol(-1) in buffer (pH 10) was estimated. Kinetic-unfolding experiments showed that the polypeptide unfolding correlated with rearrangement of the iron-sulfur centres to new ones which had strong absorption maxima at 520 and 610 nm. These new, possibly linear three-iron, clusters were coordinated to the unfolded protein but degraded slowly. From thermal experiments in the presence of GuHCl we estimated the melting temperature for the Acidianus ambivalens ferredoxin in buffer (at pH 7) to be 122 degrees C. Possible structural properties that contribute to the large thermal stability of the Acidianus ambivalens ferredoxin are discussed using a three-dimensional protein model.     

Different unfolding pathways for mesophilic and thermophilic homologues of serine hydroxymethyltransferase

To determine how much information can be transferred from folding and unfolding studies of one protein to another member of the same family or between the mesophilic and thermophilic homologues of a protein, we have characterized the equilibrium unfolding process of the dimeric enzyme serine hydroxymethyltransferase (SHMT) from two sources, Bacillus subtilis (bsSHMT) and Bacillus stearothermophilus (bstSHMT). Although the sequences of the two enzymes are highly identical ( approximately 77%) and homologous (89%), bstSHMT shows a significantly higher stability against both thermal and urea denaturation than bsSHMT. The GdmCl-induced unfolding of bsSHMT was found to be a two-step process with dissociation of the native dimer, resulting in stabilization of a monomeric species, followed by the unfolding of the monomeric species. A similar unfolding pathway has been reported for Escherichia coli aspartate aminotransferase, a member of the type I fold family of PLP binding enzymes such as SHMT, the sequence of which is only slightly identical ( approximately 14%) with that of SHMT. In contrast, for bstSHMT, a highly cooperative unfolding without stabilization of any monomeric intermediate was observed. These studies suggest that mesophilic proteins of the same structural family even sharing a low level of sequence identity may follow a common unfolding mechanism, whereas the mesophilic and thermophilic homologues of the same protein despite having a high degree of sequence identity may follow significantly different unfolding mechanisms.     

Cold instability of aponeocarzinostatin and its stabilization by labile chromophore

The conformational stability of aponeocarzinostatin, an all-beta-sheet protein with 113 amino-acid residues, is investigated by thermal-induced equilibrium unfolding between pH 2.0 and 10.0 with and without urea. At room temperature, the protein is stable in a pH range of 4.0-10.0, whereas the stability of the protein drastically decreases below pH 4.0. The thermal unfolding of aponeocarzinostatin is reversible and follows a two-state mechanism. By two-dimensional unfolding studies, the enthalpy change, heat capacity change, and free energy change for unfolding of the protein are estimated. Circular dichroism profiles suggest that this protein undergoes both heat- and cold-induced unfolding. The ellipticity changes at far- and near-UV circular dichroism suggest that the tertiary structure is disrupted but the secondary structure remains folded at low temperatures. Interestingly, the labile enediyne chromophore, which is highly stabilized by the protein, is able to protect the protein against cold-induced unfolding, but not the heat-induced unfolding.     

pH dependence thermal stability of a chymotrypsin inhibitor from Schizolobium parahyba seeds

The thermal stability of a Schizolobium parahyba chymotrypsin inhibitor (SPCI) as a function of pH has been investigated using fluorescence, circular dichroism, and differential scanning calorimetry (DSC). The thermodynamic parameters derived from all methods are remarkably similar and strongly suggest the high stability of SPCI under a wide range of pH. The transition temperature (T(m)) values ranging from 57 to 85.3 degrees C at acidic, neutral, and alkaline pH are in good agreement with proteins from mesophilic and thermophilic organisms and corroborate previous data regarding the thermal stability of SPCI. All methods gave transitions curves adequately fitted to a two-state model of the unfolding process as judged by the cooperative ratio between the van't Hoff and the calorimetric enthalpy energies close to unity in all of the pH conditions analyzed, except at pH 3.0. Thermodynamic analysis using all these methods reveals that SPCI is thermally a highly stable protein, over the wide range of pH from 3.0 to 8.8, exhibiting high stability in the pH region of 5.0-7.0. The corresponding maximum stabilities, DeltaG(25), were obtained at pH 7.0 with values of 15.4 kcal mol(-1) (combined fluorescence and circular dichroism data), and 15.1 kcal mol(-1) (DSC), considering a DeltaC(p) of 1.72 +/- 0.24 kcal mol(-1) K(-1). The low histidine content ( approximately 1.7%) and the high acidic residue content ( approximately 22.5%) suggests a flat pH dependence of thermal stability in the region 2.0-8.8 and that the decrease in thermal stability at low pH can be due to the differences in pK values of the acidic groups.     

Unfolding energetics and conformational stability of DLC8 monomer

To understand the rules governing the protein folding process it is essential to study the stability and unfolding of small monomeric proteins. Here, I present the pH dependent thermal unfolding energetics and conformational stability analysis of monomeric Dynein light chain protein (DLC8) in the pH range 3.5-2.0. DLC8 is the smallest and the most conserved light chain among the light chains of the dynein motor assembly. Thermal unfolding of DLC8 monomer is much complex with the presence of transient intermediates, which is in contrast to the notion that small proteins unfold via simple two-state process. The unfolding seems to be more cooperative at lower pH and the temperature of highest conformational stability (T(s)) is found to be maximum (295.7 K) at pH 2.76. Stability curves have been simulated to understand the thermodynamic parameters that govern the shapes of the experimentally obtained curves. Further, an effort has been made to correlate the observed differences in the denaturation energetics with the protein sequence in order to throw light on the structure-folding paradigm of the DLC8 monomer.     

Comparative thermodynamic elucidation of the structural stability of thermophilic proteins

Differential scanning calorimetry, circular dichroism, and visible absorption spectrophotometry were employed to elucidate the structural stability of thermophilic phycocyanin derived from Cyanidium caldarium, a eucaryotic organism which contains a nucleus, grown in acidic conditions (pH 3.4) at 54°C. The obtained results were compared with those previously reported for thermophilic phycocyanin derived from Synechococcus lividus, a procaryote containing no organized nucleus, grown in alkaline conditions (pH 8.5) at 52°C. The temperature of thermal unfolding (t_d) was found to be comparable between C. caldarium (73°C) and S. lividus (74°C) phycocyanins. The apparent free energy of unfolding (ΔG_[urea]=0) at zero denaturant (urea) concentration was also comparable: 9.1 and 8.7 kcal/mole for unfolding the chromophore part of the protein, and 5.0 and 4.3 kcal/mole for unfolding the apoprotein part of the protein, respectively. These values of t_d and ΔG_[urea]=0 were significantly higher than those previously reported for mesophilic Phormidium luridum phycocyanin (grown at 25°C). These findings revealed that relatively higher values of t_d and ΔG_[urea]=0 were characteristics of thermophilic proteins. In contrast, the enthalpies of completed unfolding (ΔH_d) and the half-completed unfolding (ΔH_d)1/2 for C. caldarium phycocyanin were much lower than those for S. lividus protein (89 versus 180 kcal/mole and 62 versus 115 kcal/mole, respectively). Factors contributing to a lower ΔH_d in C. caldarium protein and the role of charged groups in enhancing the stability of thermophilic proteins were discusse.     

Thermodynamic stability of a kappaI immunoglobulin light chain: relevance to multiple myeloma

Immunoglobulin light chains have two similar domains, each with a hydrophobic core surrounded by beta-sheet layers, and a highly conserved disulfide bond. Differential scanning calorimetry and circular dichroism were used to study the folding and stability of MM-kappaI, an Ig LC of kappaI subtype purified from the urine of a multiple myeloma patient. The complete primary structure of MM-kappaI was determined by Edman sequence analysis and mass spectrometry. The protein was found to contain a cysteinyl post-translational modification at Cys(214). Protein stability and conformation of MM-kappaI as a function of temperature or denaturant conditions at pH 7.4 and 4.8 were investigated. At pH 4.8, calorimetry demonstrated that MM-kappaI undergoes an incomplete, cooperative, partially reversible thermal unfolding with increased unfolding temperature and calorimetric enthalpy as compared to pH 7.4. Secondary and tertiary structural analyses provided evidence to support the presence of unfolding intermediates. Chemical denaturation resulted in more extensive protein unfolding. The stability of MM-kappaI was reduced and protein unfolding was irreversible at pH 4.8, thus suggesting that different pathways are utilized in thermal and chemical unfolding.     

High stability of a ferredoxin from the hyperthermophilic archaeon A. ambivalens: involvement of electrostatic interactions and cofactors

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (T(m)) of 122 degrees C (pH 7). To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs. Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (DeltaH[T(m)]) and T(m) from which an upper limit for the heat capacity of unfolding (DeltaC(P)) was determined to be 3.15 +/- 0.1 kJ/(mole * K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20 degrees C) observed ([GuSCN](1/2) = 3.1 M; DeltaG(U)[H(2)O] = 79 +/- 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, T(m) is 64 +/- 1 degrees C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (DeltaG(U)[H(2)O] = 20 +/- 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron-sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron-sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation.     

Thermal unfolding studies of a phytocyanin

The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at approximately 600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.4 and 68.8 degrees C at pH 7.5 and between 50.7 and 57.4 degrees C at pH 9.8. The calorimetric data indicates that at both pH values the thermally induced transition of the protein between the native and denaturated states can be described in terms of the classical Lumry-Eyring unfolding model Native<-->Unfolded-->Final. The analysis of the reversible step in the unfolding pathway demonstrates a significant reduction in conformational stability (DeltaG) of the alkaline form of the protein. Such a reduction is consistent with an enhanced flexibility of UMC at high pH and has mainly entropic character.     

Slow unfolding explains high stability of thermostable ferredoxins: common mechanism governing thermostability?

The role of kinetics in governing exceptional stability of thermophilic ferredoxins has been explored. Strikingly, unfolding-rate constants (pH 7, 20 degrees C) are over eight (log(10)) orders of magnitude slower for the thermophiles than for a large set of unrelated mesophilic proteins. Also at low pH, where ionic interactions are diminished, unfolding of the thermophilic ferredoxins is significantly slower than unfolding of the mesophiles at pH 7, emphasizing the importance of hydrophobic interactions. A kinetic barrier towards unfolding may be a common strategy used by many proteins to withstand extreme conditions.     

Unfolding by temperature and guanidine hydrochloride of chicken pancreatic polypeptide

Thermal unfolding of chicken pancreatic polypeptide at two different concentrations was studied at various pH values. The thermal stability was higher at higher protein concentrations. The transition temperatures at two different protein concentrations changed with pH in parallel and decreased by about 30 degrees C on lowering pH from 5 to 2. The results on the thermal unfolding were analyzed by assuming that the dimerization constant is independent of pH, that the thermal unfolding occurs only after the pancreatic polypeptide dimers dissociated into the monomers, and that one ionizable group participates in the acid unfolding of the monomer. The free energy change for the unfolding of the pancreatic polypeptide monomer was estimated to be 1.4 kcal/mol. The unfolding of pancreatic polypeptide by guanidine hydrochloride at pH 6.0 and 25 degrees C was also studied. The stability to guanidine hydrochloride was higher at higher protein concentrations.     

Thermostability of proteins: role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus

The dinuclear copper center (TtCuA) forming the electron entry site in the subunit II of the cytochrome c oxidase in Thermus thermophilus shows high stability toward thermal as well as denaturant-induced unfolding of the protein at ambient pH. We have studied the effect of pH on the stability of the holo-protein as well as of the apo-protein by UV-visible absorption, far-UV, and visible circular dichroism spectroscopy. The results show that the holo-protein both in the native mixed-valence state as well as in the reduced state of the metal ions and the apo-protein of TtCuA were extremely stable toward unfolding by guanidine hydrochloride at ambient pH. The thermal unfolding studies at different values of pH suggested that decreasing pH had almost no effect on the thermal stability of the protein in the absence of the denaturant. However, the stability of the proteins in presence of the denaturant was considerably decreased on lowering the pH. Moreover, the stability of the holo-protein in the reduced state of the metal ion was found to be lower than that in the mixed-valence state at the same pH. The denaturant-induced unfolding of the protein at different values of pH was analyzed using a two-state unfolding model. The values of the free energy of unfolding were found to increase with pH. The holo-protein showed that the variation of the unfolding free energy was associated with a pKa of approximately 5.5. This is consistent with the model that the protonation of a histidine residue may be responsible for the decrease in the stability of the holo-protein at low pH. The results were interpreted in the light of the reported crystal structure of the protein.     

High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low DeltaC(p) of unfolding.

To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three-state model for E.coli IPMDH and with a two-state model for T.thermophilus IPMDH, to obtain the free energy change DeltaG degrees of each unfolding process. Other thermodynamic parameters, enthalpy change DeltaH, entropy change DeltaS and heat capacity change DeltaC(p), were derived from the temperature dependence of DeltaG degrees. The main feature of the thermophilic enzyme was its lower dependence of DeltaG degrees on temperature resulting from a low DeltaC(p). The thermophilic IPMDH had a significantly lower DeltaC(p), 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low DeltaC(p) of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area DeltaASA. The results suggested that there is a large structural difference between the unfolded state of T.thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T.thermophilus IPMDH was the increase in the most stable temperature T(s). The DeltaG degrees maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature T(m) is not necessarily accompanied by a higher DeltaG degrees maximum.