Two-state Transition Model Research Articles

Structure and Dynamics of Ribonuclease A during Thermal Unfolding: The Failure of the Zimm Model.

Disordered regions as found in intrinsically disordered proteins (IDP) or during protein folding define response time to stimuli and protein folding times. Neutron spin-echo spectroscopy is a powerful tool to directly access the collective motions of the unfolded chain to enlighten the physical origin of basic conformational relaxation. During the thermal unfolding of native ribonuclease A, we examine the structure and dynamics of the disordered state within a two-state transition model using polymer models, including internal friction, to describe the chain dynamics. The presence of four disulfide bonds alters the disordered configuration to a more compact configuration compared to a Gaussian chain that is defined by the additional links, as demonstrated by coarse-grained simulation. The dynamics of the disordered chain is described by Zimm dynamics with internal friction (ZIF) between neighboring amino acids. Relaxation times are dominated by mode-independent internal friction. Internal friction relaxation times show an Arrhenius-like behavior with an activation energy of 33 kJ/mol. The Zimm dynamics is dominated by internal friction and suggest that the characteristic motions correspond to overdamped elastic modes similar to the motions observed for folded proteins but within a pool of disordered configurations spanning the configurational space. For IDP, internal friction dominates while solvent friction and hydrodynamic interactions are smaller corrections.

Effects of Sequence Changes on the Torsion Elastic Constant and Persistence Length of DNA. Applications of the Two-State Model.

Previous experimental studies of the effects of changes in sequence on the elastic constants and other properties of DNAs are analyzed using the recently parametrized two-state cooperative transition model. With appropriate assumptions regarding the relative preference of particular subsequences for the b and a states, the model gives an approximately quantitative account of the surprisingly large observed effects of replacing 25 bp of natural sequence near the middle of an ∼1100 bp DNA by 16 bp of alternating (CG)8 sequence on the fraction fb of base-pairs in the b-state and on the torsion elastic constant. This demonstrates the effect of the strong cooperativity of the transition to significantly influence the state of the DNA over surprisingly large domains of the flanking DNA. With appropriate assumptions regarding the disposition of nonrelaxing (permanent) and slowly relaxing bends between the a and b states, the two-state model resolves the discrepancy between experimental estimates of the magnitude of the nonrelaxing bends obtained from studies of synthetic straight and natural DNA sequences by cryo-electron microscopy on the one hand and by j-factors inferred from the kinetics of ligase cyclization of small ∼200 bp circular DNAs on the other. Approximately quantitative agreement with the experiments is again obtained.

Temperature-dependence of the bending elastic constant of DNA and extension of the two-state model. Tests and new insights

Review and analyses of the experimental data indicate that in nearly all cases bending elastic constants of the effective springs between bp of DNA actually undergo a net increase with increasing T from 278 to 315 K. The exceptions to this rule are bending elastic constants obtained from equilibrium topoisomer distributions of a 2686 bp pUC19 DNA by assuming a fixed T-independent value of the torsion elastic constant. When the same data are analyzed using measured T-dependent values of the torsion elastic constant, which decline with increasing T, a modest increase in bending elastic constant with increasing T is obtained. After revising the torsion elastic constants of the previously formulated two-state cooperative transition model to account for additional data, that model is fitted to the bending elastic constants reckoned from the aforementioned topoisomer distributions to determine the best-fit values for each state. The rather good fit implies a strong negative linear correlation between the inverse bending and inverse torsion elastic constants as T is varied. Predictions of the resulting two-state model, wherein each state has fixed bending and torsion elastic constants, agree surprisingly well with single-molecule relative extension and torque data. The same model also yields good agreement with numerous other experimental data. With increasing T the equilibrium is shifted from the (longer, torsionally stiffer, flexurally softer) b-state toward the (shorter, torsionally softer, flexurally stiffer) a-state. This transition is suggested to be the origin of the so-called broad pre-melting transition exhibited by many, but not all, DNAs.

Denaturation properties and folding transition states of leghemoglobin and other heme proteins.

This work reports unfolding transitions of monomeric heme proteins leghemoglobin (Lb), myoglobin (Mb), and cytochrome c (Cyt c) utilizing UV-Vis spectral and steady-state and time-resolved fluorescence methods. Conformational stabilities of the native "folded" state of the proteins and their "unfolded" states were investigated in the light of a two-state transition model. Two-state transition values for ΔGD (298K) were obtained by denaturation with the chaotropic agents urea and guanidium hydrochloride (GdnHCl). The free energy value of Lb is the lowest compared to Cyt c and Mb along the denaturation pathway. The m value is also the lowest for Lb compared to Cyt c and Mb. The m value (a measure of dependence of ΔGD on denaturant concentration) for Cyt c and Mb is lower when it is denatured with urea compared to GdnHCl. The UV-Vis absorbance maximum and steady state fluorescence emission maximum were drastically red shifted in the presence of a certain denaturant concentration both in cases of Mb and Lb, but the scenario is different for Cyt c. The results are analyzed using a two-state transition model. The lifetime data clearly indicate the presence of an intermediate state during denaturation. The unfolding transition can modulate the conformation, stability, and surface exposure of these biologically important proteins.

Calorimetric Behavior of Phosphatidylcholine/Phosphatidylethanolamine Bilayers is Compatible with the Superlattice Model

Differential scanning calorimetry was used to study the phase behavior of binary lipid bilayers consisting of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying acyl chain length. A two-state transition model was used to resolve the individual transition components, and the two-state transition enthalpy, the relative enthalpy, and the transition temperature of each component were plotted as a function of composition. Intriguingly, abrupt changes in these thermodynamic parameters were observed at or close to many "critical" X(PE) values predicted by the superlattice model proposing that phospholipids with different headgroups tend to adopt regular rather than random lateral distributions. Statistical analysis indicated that the agreement between the observed and predicted "critical" compositions is highly significant. Accordingly, these data provide strong evidence that the molecules in PC/PE bilayers tend to adopt regular, superlattice-like lateral arrangements, which could be involved in the regulation of the lipid compositions of biological membranes.

Cross-Strand Coupling and Site-Specific Unfolding Thermodynamics of a Trpzip β-Hairpin Peptide Using 13C Isotopic Labeling and IR Spectroscopy

Conformational properties of a 12-residue tryptophan zipper (trpzip) beta-hairpin peptide (AWAWENGKWAWK-NH(2), a modification of the original trpzip2 sequence) are analyzed under equilibrium conditions using ECD and IR spectra of a series of variants, singly and doubly C(1)-labeled with (13)C on the amide CO. The characteristic features of the (13)CO component of the amide I' IR band and their sensitivity to the local structure of the peptide are used to differentiate stabilities for parts of the hairpin structure. Doubly labeled peptide spectra indicate that the ends of the beta-strands are frayed and that the center part is more stable as would be expected from formation of a stable hydrophobic core consisting of four tryptophan residues, and supported by MD simulations. NMR analyses were used to determine a best fit solution structure that is in close agreement with that of trpzip2, except for a small variation in the turn geometry. The distinct vibrational coupling patterns of the labeled sites based on this structure are also well matched by ab initio DFT-level calculations of their IR spectral patterns. Thermal unfolding of the peptides as studied with CD spectra could be fit with an apparent two-state transition model. ECD senses only the tryptophan interactions (tertiary-like) and their overall environment, as shown by TD-DFT modeling of the Trp-Trp pi-pi ECD. However, variation of the amide I IR spectra of (13)C-isotopomers showed that the thermal unfolding process is not cooperative in terms of the peptide backbone (secondary structure), since the transition temperatures sensed for labeled modes differ from those for the whole peptide. The thermal data also evidence dependence on concentration and pH but these cause little spectral variation. This study illustrates the consequences of multistate conformational change at the residue- or sequence-specific level in a system whose structure is dominated by hydrophobic collapse.

Comparison of the chemical and thermal denaturation of proteins by a two-state transition model

The conformational stabilities of eight proteins in terms of the free energy differences between the native “folded” state of the protein and its “unfolded” state were determined at 298 K by two methods: chemical denaturation at 298 K and extrapolation to 298 K of the thermal denaturation results at high temperature. The proteins were expressed in Escherichia coli from the Haemophilus influenzae and E. coli genes at different levels of expression, covered a molecular mass range from 13 to 37 kg mol −1 per monomeric unit (some exhibiting unique structural features), and were oligomeric up to four subunits. The free energy differences were determined by application of a two-state transition model to the chemical and thermal denaturation results, ranged from 9.4 to 148 kJ mol −1 at 298 K, and were found to be within the experimental uncertainties of both methods for all of the proteins. Any contributions from intermediate states detectable from chemical and thermal denaturation differences in the unfolding free energy differences in these proteins are within the experimental uncertainties of both methods.

Helicity propensity and interaction of synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H: A circular dichroism study

The secondary structure of two synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H was determined by circular dichroism. In particular, the propensity of these peptides to assume an ordered structure was investigated upon by changing the solvent's polarity and the temperature. A reduction of solvent polarity led to a significant increase in the α-helix content in the case of HR1, whereas only a slight change in the secondary structure was observed in the case of HR2. In both cases the conformational change followed a two-state transition model. The interaction of the peptides was monitored by the conformational change in the mixture with respect to the single peptides. However, formation of the complex did not significantly enhance thermal stability. A reliable estimation of the secondary structure was obtained by optimising the experimental conditions to collect CD data down to 180 nm, and by comparing the structure data yielded by different software packages.

Cooperative unfolding of Escherichia coli ribosome recycling factor originating from its domain–domain interaction and its implication for function

Cooperative unfolding of Escherichia coli ribosome recycling factor (RRF) and its implication for function were investigated by comparing the in vitro unfolding and the in vivo activity of wild-type E. coli RRF and its temperature-sensitive mutant RRF V117D. The experiments show that mutation V117D at domain I could perturb the domain II structure as evidenced in the near-UV CD and tyrosine fluorescence spectra though no significant globular conformation change occurred. Both equilibrium unfolding induced by heat or denaturant and kinetic unfolding induced by denaturant obey the two-state transition model, indicating V117D mutation does not perturb the efficient interdomain interaction, which results in cooperative unfolding of the RRF protein. However, the mutation significantly destabilizes the E. coli RRF protein, moving the thermal unfolding transition temperature range from 50–65 to 35–50 °C, which spans the non-permissive temperature for the growth of E. coli LJ14 strain ( frr ts). The in vivo activity assays showed that although V117D mutation results in a temperature sensitive phenotype of E. coli LJ14 strain ( frr ts), over-expression of mutant RRF V117D can eliminate the temperature sensitive phenotype at the non-permissive temperature (42 °C). Taking all the results into consideration, it can be suggested that the mechanism of the temperature sensitive phenotype of the E. coli LJ14 cells is due to inactivation of mutant RRF V117D caused by unfolding at the non-permissive temperatures.

Single Giant Unilamellar Vesicle Method Reveals Effect of Antimicrobial Peptide Magainin 2 on Membrane Permeability

It is thought that magainin 2, an antimicrobial peptide, acts by binding to lipid membranes. Recent studies using a suspension of large unilamellar vesicles (LUVs) indicate that magainin 2 causes gradual leakage from LUVs containing negatively charged lipids. However, the details of the characteristics of the membrane permeability and the mechanism of pore formation remain unclear. In this report, we investigated the interaction of magainin 2 with single giant unilamellar vesicles (GUVs) composed of a dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol mixture (50% DOPG/50% DOPC GUVs) containing the fluorescent dye, calcein, by phase contrast, fluorescence microscopy using the single GUV method. Low concentrations (3-10 microM) of magainin 2 caused the rapid leakage of calcein from single GUVs but did not disrupt the liposomes or change the membrane structure, showing directly that magainin 2 forms membrane pores through which calcein leaked. The rapid leakage of calcein from a GUV started stochastically, and once it began, the complete leakage occurred rapidly (6-60 s). The fraction of completely leaked GUV, P(L), increased with time and also with an increase in magainin 2 concentration. Shape changes in these GUVs occurred prior to the pore formation and also at lower concentrations of magainin 2, which could not induce the pore formation. Their analysis indicates that binding of magainin 2 to the external monolayer of the GUV increases its membrane area, thereby raising its surface pressure. The addition of lysophosphatidylcholine into the external monolayer of GUVs increased P(L). On the basis of these results, we propose the two-state transition model for the pore formation.

Thermal Aggregation of Methyl Cellulose in Aqueous Solution: A Thermodynamic Study and Protein Partitioning Behaviour

The formation of a biphasic system from aqueous solutions of methyl cellulose induced by temperature was studied through heating curves of the polymer solution measured by absorbance spectroscopy, differential scanning calorimetry and solution viscosimetry. The treatment of the heating curve data according to a reversible two-state transition model allows us to calculate the middle point temperature (Tm) of the formation of the two phases and the thermodynamic functions associated to the polymer aggregation. The middle point temperature was found within the range 50–70 °C. It decreased significantly in a Na2SO4 0.3 M medium when the polymer concentration increased. The heat associated to the two-phase formation was positive and it increased with increases in temperature. Cosolutes that affect the water structure induced changes in the Tm values, which suggests the presence of a hydrophobic effect in the two-phase formation from the polymer solution. Hydrophilic proteins were partitioned in favour of the methyl cellulose rich phase according to their surface hydrophobicity. The partition was also influenced by the presence of salts that modify the protein hydrophobicity such as sodium sulphate.

Raf RBD and ubiquitin proteins share similar folds, folding rates and mechanisms despite having unrelated amino acid sequences.

Recent experimental and theoretical studies in protein folding suggest that the rates and underlying mechanisms by which proteins attain the native state are largely determined by the topological complexity of a specific fold rather than by the fine details of the amino acid sequences. However, such arguments are based upon the examination of a limited number of protein folds. To test this view, we sought to investigate whether proteins belonging to the ubiquitin superfamily display similar folding behavior. To do so, we compared the folding-unfolding transitions of mammalian ubiquitin (mUbi) with those of its close yeast homologue (yUbi), and to those of the structurally related Ras binding domain (RBD) of the serine/threonine kinase raf that displays no apparent sequence homology with the ubiquitin family members. As demonstrated for mUbi [Krantz, B. A., and Sosnick, T. R. (2000) Biochemistry 39, 11696-11701], we show that a two-state transition model with no burst phase intermediate can describe folding of both yUbi and raf RBD. We further demonstrate that (1) all three proteins refold at rates that are within 1 order of magnitude (1800, 1100, and 370 s(-1) for mUbi, raf RBD, and yUbi, respectively), (2) both mUbi and raf RBD display similar refolding heterogeneity, and (3) the folding free energy barriers of both mUbi and raf RBD display a similar temperature dependence and sensitivity to a stabilizing agent or to mutations of a structurally equivalent central core residue. These findings are consistent with the view that rates and mechanisms for protein folding depend mostly on the complexity of the native structure topology rather than on the fine details of the amino acid sequence.

Thermal unfolding of ribonuclease T1 studied by multi-dimensional NMR spectroscopy

Thermal unfolding of ribonculease (RNase) T1 was studied by 1H nuclear Overhauser enhancement spectroscopy (NOESY) and 1H- 15N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy at various temperatures. Native RNase T1 is a single-chain molecule of 104 amino acid residues, and has a single alpha-helix and two beta-sheets, A and B, which consist of two and five strands, respectively. Singular value decomposition analysis based on temperature-dependent HSQC spectra revealed that the thermal unfolding of RNase T1 can be described by a two-state transition model. The midpoint temperature and the change in enthalpy were determined as 54.0 degrees C and 696 kJ/mol, respectively, which are consistent with results obtained by other methods. To analyze the transition profile in more detail, we investigated local structural changes using temperature-dependent NOE intensities. The results indicate that the helical region starts to unfold at lower temperature than some beta-strands (B3, B4, and B5 in beta-sheet B). These beta-strands correspond to the hydrophobic cluster region, which had been expected to be a folding core. This was confirmed by structure calculations using the residual NOEs observed at 56 degrees C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.

Thermal denaturation of spinach plastocyanin: effect of copper site oxidation state and molecular oxygen.

The thermal denaturation of the cupredoxin plastocyanin (PC) from spinach has been studied with the aim of improving the understanding of factors involved in the conformational stability of antiparallel beta-sheet proteins. Studies using differential scanning calorimetry have been complemented with nuclear magnetic resonance spectroscopy, absorbance spectroscopy, dynamic light scattering, and mass spectrometry in elucidation of the effect of the copper-site oxidation state on the irreversible thermal denaturation process. Our results indicate that copper-catalyzed oxidation of the metal-ligating cysteine is the sole factor resulting in thermal irreversibility. However, this can be prevented in reduced protein by the removal of molecular oxygen. Application of a two-state equilibrium transition model to the folding process thus allowed the extraction of thermodynamic parameters for the reduced protein (Delta(trs)H = 494 kJ mol(-1), DeltaH(vH) = 343 kJ mol(-1), and T(m) = 71 degrees C). However, anaerobically denatured oxidized protein and all aerobically denatured species undergo covalent modification as a result of the copper-catalyzed oxidation of the metal-ligating cysteine residue resulting in the formation of both oxidized monomers and disulfide-linked dimers. On the basis of these results, a general mechanism for the irreversible thermal denaturation of cupredoxins is proposed. The results presented here also indicate that PC, as opposed to the previously characterized homologous protein azurin, unfolds via at least one significantly populated intermediate state (DeltaH(vH)/Delta(trs)H = 0.7) despite the almost identical native state topologies of these proteins. These findings will aid the characterization of the stability of PC and other cupredoxins and possibly of all cysteine-ligating metal-binding proteins.

Characterization of folding pathways of the type-1 and type-2 periplasmic binding proteins MglB and ArgT.

The family of periplasmic binding proteins (PBPs) is believed to have arisen from a common ancestor and to have differentiated into two types. At first approximation, both types of PBPs have the same fold pattern, reflecting their common origin. However, the connection between the main chains of a type 2 PBP is more complicated than a type 1 PBP's. We have been interested in the possibility that such structural changes affect the folding of PBPs. In this study, we have characterized the folding pathways of MglB (a type 1 PBP) and ArgT (a type 2 PBP) by using urea gradient gel electrophoresis, fast protein size-exclusion liquid chromatography and hydrophobic dye ANS binding assay. We found a distinct difference in folding between these two proteins. The folding of MglB followed a simple two-state transition model, whereas the folding of ArgT was more complicated.

STATISTICAL MECHANICAL FOUNDATION FOR THE TWO-STATE TRANSITION IN PROTEIN FOLDING OF SMALL GLOBULAR PROTEINS

We discuss the statistical mechanical foundation for the two-state transition in the protein folding of small globular proteins. In the standard arguments of protein folding, the statistical search for the ground state is carried out from astronomically many conformations in the configuration space. This leads us to the famous Levinthal's paradox. To resolve the paradox, Gō first postulated that the two-state transition — all-or-none type transition — is very crucial for the protein folding of small globular proteins and used the Gō's lattice model to show the two-state transition nature. Recently, there have been accumulated many experimental results that support the two-state transition for small globular proteins. Stimulated by such recent experiments, Zwanzig has introduced a minimal statistical mechanical model that exhibits the two-state transition. Also, Finkelstein and coworkers have discussed the solution of the paradox by considering the sequential folding of a small globular protein. On the other hand, recently Iguchi have introduced a toy model of protein folding using the Rubik's magic snake model, in which all folded structures are exactly known and mathematically represented in terms of the four types of conformations: cis-, trans-, left and right gauche-configurations between the unit polyhedrons. In this paper, we study the relationship between the Gō's two-state transition, the Zwanzig's statistical mechanics model and the Finkelsteinapos;s sequential folding model by applying them to the Rubik's magic snake models. We show that the foundation of the Gō's two-state transition model relies on the search within the equienergy surface that is labeled by the contact order of the hydrophobic condensation. This idea reproduces the Zwanzig's statistical model as a special case, realizes the Finkelstein's sequential folding model and fits together to understand the nature of the two-state transition of a small globular protein by calculating the physical quantities such as the free energy, the contact order and the specific heat. We point out the similarity between the liquid-gas transition in statistical mechanics and the two-state transition of protein folding. We also study morphology of the Rubik's magic snake models to give a prototype model for understanding the differences between α-helices proteins and β-sheets proteins.

Measurement and analysis of results obtained on biological substances with d.s.c.

The differential scanning calorimeter (d.s.c.) has been widely used to determine the thermodynamics of phase transitions and conformational changes in biological systems including proteins, nucleic acid sequences, and lipid assemblies. The d.s.c monitors the temperature difference between two vessels, one containing the biological solution and the other containing a reference solution, as a function of temperature at a given scan rate. Recommendations for d.s.c. measurement procedures, calibration procedures, and procedures for testing the performance of the d.s.c. are described. Analysis of the measurements should include a correction for the time response of the instrument and conversion of the power versus time curve to a heat capacity versus temperature plot. Thermodynamic transition models should only be applied to the analysis of the heat capacity curves if the model-derived transition temperatures and enthalpies are independent of the d.s.c. scan rate. Otherwise, kinetic models should be applied to the analysis of the data. Application of thermodynamic transition models involving two states, two states and dissociation, and three states to the heat capacity versus temperature data are described. To check the operating performance with standard d.s.c.s, samples of (1 to 10) mg · cm3solutions of hen egg white lysozyme in 0.1 mol · dm−3HCl–glycine buffer at pH = (2.4 ± 0.1) were sent to six different d.s.c. laboratories worldwide. The values obtained from proper measurements and application of a two-state transition model yielded an average unfolding transition temperature for lysozyme of 331.2 K with values ranging between T= 329.4 K and 331.9 K, and an average transition enthalpy of 405 kJ · mol−1with values ranging from 377 kJ · mol−1to 439 kJ · mol−1. It is recommended that the reporting of d.s.c. results be specific with regard to the composition of the solution, the operating conditions and calibrations of the d.s.c. determination of base lines that may be model dependent, and the model used in the analysis of the data.

Pressure effect on denaturant-induced unfolding of hen egg white lysozyme.

The influence of hydrostatic pressure (< or =100 MPa) on denaturant-induced unfolding of hen egg white lysozyme was investigated by means of ultraviolet spectroscopy at various temperatures. Assuming a two-state transition model, the dependence of Gibbs free-energy change of unfolding on the denaturant concentration was calculated. Under applied hydrostatic pressure, these data were interpreted as suggesting that a two-state model is not applicable in a restricted temperature range; the dominant effect of hydrostatic pressure is to affect the cooperativity in protein unfolding due to a chemical equilibrium shift in the direction of the reduction in the system volume. The deviation from the two-state transition model appears to be rationalized by assuming that applied pressure induces an intermediate conformation between the native and unfolded states of the protein. The implication of the thermodynamic stability of protein under pressure was discussed.

Measurement and analysis of results obtained on biological substances with differential scanning calorimetry (IUPAC Technical Report)

Abstract Differential scanning calorimeters (DSCs) have been widely used to determine the thermodynamics of phase transitions and conformational changes in biological systems including proteins, nucleic acid sequences, and lipid assemblies. DSCs monitor the temperature difference between two vessels, one containing the biological solution and the other containing a reference solution, as a function of temperature at a given scan rate. Recommendations for DSC measurement procedures, calibration procedures, and procedures for testing the performance of the DSC are described. Analysis of the measurements should include a correction for the time response of the instrument and conversion of the power vs. time curve to a heat capacity vs. temperature plot. Thermodynamic transition models should only be applied to the analysis of the heat capacity curves if the model-derived transition temperatures and enthalpies are independent of the DSC scan rate. Otherwise, kinetic models should be applied to the analysis of the data. Application of thermodynamic transition models involving two states, two states and dissociation, and three states to the heat capacity vs. temperature data are described. To check the operating performance with standard DSCs, samples of 1 to 10 mg mL­1 solutions of hen egg white lysozyme in 0.1 M HCl-glycine buffer at pH = 2.4 ± 0.1 were sent to six different DSC laboratories worldwide. The values obtained from proper measurements and application of a two-state transition model yielded an average unfolding transition temperature for lysozyme of 331.2 K with values ranging from 329.4 to 331.9 K, and an average transition enthalpy of 405 kJ mol­1 with values ranging from 377 to 439 kJ mol­1. It is recommended that the reporting of DSC results be specific with regard to the composition of the solution, the operating conditions and calibrations of the DSC, determination of base lines that may be model-dependent, and the model used in the analysis of the data.

Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry.

Thermodynamics of the thermal dissociation transitions of 10 bp PNA/DNA duplexes and their corresponding DNA/DNA duplexes in 10 mM sodium phosphate buffer (pH 7.0) were determined from differential scanning calorimetry (DSC) measurements. The PNA/DNA transition temperatures ranged from 329 to 343 K and the calorimetric transition enthalpies ranged from 209 +/- 6 to 283 +/- 37 kJ mol(-1). The corresponding DNA/DNA transition temperatures were 7-20 K lower and the transition enthalpies ranged from 72 +/- 29 to 236 +/- 24 kJ mol(-1). Agreement between the DSC and UV monitored melting (UVM) determined transition enthalpies validated analyzing the UVM transitions in terms of a two-state transition model. The transitions exhibited reversibility and were analyzed in terms of an AB = A + B two-state transition model which yielded van't Hoff enthalpies in agreement with the transition enthalpies. Extrapolation of the transition enthalpies and free energy changes to ambient temperatures yielded more negative values than those determined directly from isothermal titration calorimetry measurements on formation of the duplexes. This discrepancy was attributed to thermodynamic differences in the single-strand structures at ambient and at the transition temperatures, as indicated by UVM measurements on single DNA and PNA strands.