Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in six 22-base pair deoxyoligonucleotides.

Effects of different end sequences on stability, circular dichroism spectra (CD), and enzyme binding properties were investigated for six 22-base pair, non-self-complementary duplex DNA oligomers. The center sequences of these deoxyoligonucleotides have 8-14 base pairs in common and are flanked on both sides by sequences differing in context and A-T content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming a two-state melting transition. Melting free energies (20 degrees C) of the six DNAs evaluated from DSC experiments ranged from -18.7 to -32.7 kcal/mol. van't Hoff estimates of the free energies ranged from -18.5 to -48.0 kcal/mol. With either method, the trends in free energy as a function of sequence were identical. Equilibrium binding by BamHI restriction endonuclease to the 22-base pair DNAs was also investigated. The central eight base pairs of all six molecules, 5'-A-GGATCC-A-3', contained a BamHI recognition sequence bounded by A-T base pairs. Magnesium free binding assays were performed by titering BamHI against a constant concentration of each of the deoxyoligonucleotide substrates and analyzing reaction products by gel retardation. Binding isotherms of the total amount of bound DNA versus protein concentration were constructed which provided semiquantitative estimates of the equilibrium dissociation constants for dissociation of BamHI from the six DNA oligomers. Dissociation constants ranged from 0.5 x 10(-)(9) to 12.0 x 10(-)(9) M with corresponding binding free energies of -12.5 to -10.6 (+/-0. 1) kcal/mol. An inverse relationship is found when binding and stability are compared.     

Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain.

The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E. coli have been determined using a combination of high resolution NMR, ultracentrifugation, 15N NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry. The subunit molecular weight is 7,438 kDa., compared with 14.5+/-1.0 kDa. determined by equilibrium sedimentation, indicating a dimeric structure. The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface. The Stokes radius of the protein decreases from a high plateau value (ca. 22 A) at protein concentrations greater than 4 microM to about 18 A at concentrations less than 0.1 microM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca. 71.5 degrees C at 36 microM). The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively. The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with deltaH1=23 kJ mol(-1). K1(298)=0.4 microM and deltaH2= 184 kJ mol(-1). This is in good agreement with direct calorimetric analysis of the thermal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol(-1) and a van't Hoff enthalpy change of 354 kJ mol(-1), confirming the dimer to monomer unfolding. The thermodynamic data can be reconciled with the observed mode of dimerisation. 15N NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein. The role of dimerisation of this domain in the excision repair mechanism is discussed.     

Linked thermal and solute perturbation analysis of cooperative domain interactions in proteins. Structural stability of diphtheria toxin

The temperature and guanidine hydrochloride (GuHCl) dependence of the structural stability of diphtheria toxin has been investigated by high-sensitivity differential scanning calorimetry. In 50 mM phosphate buffer at pH 8.0 and in the absence of GuHCl, the thermal unfolding of diphtheria toxin is characterized by a transition temperature (Tm) of 54.9 degrees C, a calorimetric enthalpy change (delta H) of 295 kcal/mol, and a van't Hoff to calorimetric enthalpy ratio of 0.57. Increasing the GuHCl concentration lowers the transition temperature and the calorimetric enthalpy change. At the same time, the van't Hoff to calorimetric enthalpy ratio increases until it reaches a value of 1 at 0.3 M GuHCl and remains constant thereafter. At low GuHCl concentrations (0-0.3 M), the thermal unfolding of diphtheria toxin is characterized by the presence of two transitions corresponding to the A and B domains of the protein. At higher GuHCl concentrations (0.3-1 M), the A domain is unfolded at all temperatures, and only one transition corresponding to the B domain is observed. Under these conditions, the most stable protein conformation at low temperatures is a partially folded state in which the A domain is unfolded and the B domain folded. A general model that explicitly considers the energetics of domain interactions has been developed in order to account for the stability and cooperative behavior of diphtheria toxin. It is shown that this cooperative domain interaction model correctly accounts for the temperature location as well as the shape and area of the calorimetric curves. Under physiological conditions, domain-domain interactions account for most of the structural stability of the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation.

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wave-lengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 +/- 0.4 degrees C and the enthalpy of unfolding at this temperature (delta Hm) is 40.8 +/- 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of delta Hm versus Tm yield a mean value of 590 +/- 120 cal/(mol.K) for the change in heat capacity upon unfolding (delta Cp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 +/- 110 cal/(mol.K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining delta Cp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (delta Gu) were plotted versus temperature, the resulting curve appeared linear; therefore, delta Cp could not be determined. However, the data for delta Gu versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of delta Gu in urea are identical to values measured directly.     

Thermodynamics of a stable yeast 5.8S rRNA hairpin helix.

The 5. 8S ribosomal RNA of bakers yeast contains one particularly stable hairpin helix which is isolated by partial T1 ribonuclease digestion. Thermal hyperchromism analysis of the hairpin fragment showed that it dissociates cooperatively with 18% hyperchromism, with a Tm of 83 degree C at 2.7 mM sodium ion concentration, and with a hyperchromic difference spectrum indicative of over 90% G + C content. The probable secondary structure for the fragment was used to predict a helix free energy, delta G = -16.2 kcal/mole, which was the same as that determined from the melting equilibrium. The predicted enthalpy however, was 77% of the value, delta H = -114 kcal/mole, determined from the van't Hoff relationship. The effect on these data of a G.U base pair within the 9 base pair helix is discussed.     

Differential distortion of substrate occurs when it binds to DNA photolyase: a 2-aminopurine study

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it is exposed to ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) upon absorption of blue-green light. We have used the fluorescent adenine analogue 2-aminopurine (2Ap) to probe the local double-helical structure of the DNA substrate when it binds to the protein. Duplex melting temperatures and van't Hoff enthalpies were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and CPD on DNA stability. Steady-state fluorescence measurements of the single- and double-stranded oligos showed that the local region around the 5'-side of the CPD lesion was more disrupted and destacked than the 3'-side in substrate-protein complexes. These results were compared with those of a protein-substrate crystal structure, demonstrating that the crystal structure and solution-state studies are in agreement with regard to the differential distortions of the target DNA at the active site of the protein.     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

A new method for the determination of stability parameters of proteins from their heat-induced denaturation curves

A new method has been developed for determining the stability parameters of proteins from their heat-induced transition curves followed by observation of changes in the far-UV circular dichroism (CD). This method of analysis of the thermal denaturation curve of a protein gave values of stability parameters that not only are identical to those measured by the differential scanning calorimetry (DSC), but also are measured with the same error as that observed with a calorimeter. This conclusion has been reached from our studies of the reversible heat-induced denaturation of lysozyme and ribonuclease A at various pH values. For each protein, the conventional method of analysis of the conformational transition curve, which assumes a linear temperature dependence of the pre- and posttransition baselines, gave the estimate of DeltaH(van)(m) (enthalpy change on denaturation at T(m), the midpoint of denaturation) which is significantly lower than DeltaH(cal)(m), the value obtained from DSC measurements. However, if the analysis of the same denaturation curve assumes that a parabolic function describes the temperature dependence of the pre- and posttransition baselines, there exists an excellent agreement between DeltaH(van)(m) and DeltaH(cal)(m) of the protein. The latter analysis is supported by the far-UV CD measurements of the oxidized ribonuclease A as a function of temperature, for the temperature dependence of this optical property of the protein is indeed nonlinear. Furthermore, it has been observed that, for each protein, the constant-pressure heat capacity change (DeltaC(p)) determined from the plots of DeltaH(van)(m) versus T(m) is independent of the method of analysis of the transition curve.     

基于蛋白质二级结构热稳定性的正交方法

对蛋白质热稳定性的研究是解析蛋白高级结构,开发蛋白功能及新药物研发过程中的一个重要环节,是对其结构分析的一个重要关切点。观测蛋白质的圆二色光谱随温度程序变化而改变是研究其热稳定性的常用手段,传统的实验方法为选用某一单波长作为测试点,通过连续升温测试蛋白在单波长下的圆二色变温曲线,然后拟合出Tm值,此方法所得的信息有限,并且如何选取该单波长点是一个有争论的问题。本实验开发和优化了蛋白质二级结构热稳定性测试的一种正交方法,以牛血清蛋白及血红蛋白为研究对象,利用180-260 nm范围内的圆二色全光谱热变性测试来考察蛋白质构象随温度的变化而改变的过程。结果显示,当吸光度在合适的范围内,蛋白的热变性中点温度(Tm值)不受浓度的影响。作为对比,在180-260 nm范围内采用单波长法重复牛血清蛋白的热变性测试,结果表明,除了端点和交叉点外,其余波长的单点法的结果与全光谱正交法所测出的蛋白的Tm值是一致的,说明此两种方法在Tm值的测定上都是可靠的。以上通过对两种方法的全面对比发现,全光谱正交法不仅可以测定选定范围的每个波长的变温曲线,而且可以观测蛋白质的整体结构热变性过程,而用单波长法在同样的实验时间不能有效地记录蛋白构象在热变性过程中的变化过程。     

Refinement of noncalorimetric determination of the change in heat capacity, DeltaC(p), of protein unfolding and validation across a wide temperature range

The change in heat capacity, DeltaC(p), on protein unfolding has been usually determined by calorimetry. A noncalorimetric method which employs the Gibbs-Helmholtz relationship to determine DeltaC(p) has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which DeltaG is zero, T(m), and change in enthalpy at T(m) are determined by thermal denaturation and DeltaC(p) is then calculated using the Gibbs-Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of DeltaC(p) consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, T(m), and the van't Hoff enthalpy, DeltaH(vH), from a thermal denaturation, gives a reasonable estimate of DeltaC(p), albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs-Helmholtz equation as a function of stability and temperature while simultaneously fitting DeltaC(p), T(m), and DeltaH(vH) gives values for the last two parameters that are in excellent agreement with experimental values.     

Effect of methylamine and plasmin on the conformation of human alpha 2-macroglobulin as revealed by differential scanning calorimetric analysis.

Differential scanning calorimetric analysis was used as a probe of the conformational alteration in human alpha 2-macroglobulin (AM) upon its complex formation with methylamine and with the protease, human plasmin. The slow electrophoretic form of AM displayed a single thermal transition, characterized by a temperature midpoint (Tm) of 65.8 +/- 0.3 degrees, a calorimetric enthalpy (delta Hc) of 2,550 +/- 150 kcal/mol and a van't Hoff enthalpy (delta Hvh) of 140 kcal/mol. In the presence of sufficient methylamine to irreversibly disrupt the four thiol ester bonds in AM, a single thermal transition was obtained, characterized by a Tm of 62.8 +/- 0.3 degrees, a delta Hc of 1,700 +/- 100 kcal/mol, and a delta Hvh of 169 kcal/mol. These data suggest that a major conformational alteration is produced in AM upon complex formation with methylamine. When plasmin interacts with AM, the resulting thermogram displays Tm values for AM of 68-69 degrees and 77 degrees, also suggestive of a large conformational alteration in AM. However, this latter alteration appears dissimilar to the change induced by methylamine.     

Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.

Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein] = 1) was asymmetric about the transition temperature of 78 degrees C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 degrees C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (delta Hcal) of 200 kcal/mol. A van't Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van't Hoff enthalpies found from fits of the DSC (delta HvH = 184 kcal/mol) and spectroscopic (delta HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR in equilibrium with U+R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N in equilibrium with U). Furthermore, the NR in equilibrium with U+R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N in equilibrium with U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR in equilibrium with U+R model is the simplest model that describes the thermal unfolding of this ligand-bound protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.     

Thermodynamics of unfolding of beta-trypsin at pH 2.8

The unfolding equilibrium of beta-trypsin induced by thermal and chemical denaturation was thermodynamically characterized. Thermal unfolding equilibria were monitored using UV absorption and both far- and near-UV CD spectroscopy, while fluorescence was used to monitor urea-induced transitions. Thermal and urea transition curves are reversible and cooperative and both sets of data can be reasonably fitted using a two-state model for the unfolding of this protein. Plots of the fraction denatured, calculated from thermal denaturation curves at different wavelengths, versus temperature are coincident. In addition, the ratio of the enthalpy of denaturation obtained by scanning calorimetry to the van't Hoff enthalpy is close to unity, which supports the two-state model. Considering the differences in experimental approaches, the value for the stability of beta-trypsin estimated from spectroscopic data (deltaGu = 6.0 +/- 0.2 kcal/mol) is in reasonable agreement with the value calculated from urea titration curves (deltaGUH2O = 5.5 +/- 0.3 kcal/mol) at pH 2.8 and 300 degrees K.     

Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in four 40 base pair deoxyoligonucleotides

Effects of different end sequences on melting, circular dichroism spectra (CD), and enzyme binding properties were investigated for four 40 base pair, non-self-complementary duplex DNA oligomers. The center sequences of these oligoduplexes have either of two 22 base pair modules flanked on both sides by sequences differing in AT content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from a van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming that the melting transition is two-state. Melting free energies (20 degrees C) evaluated from DSC melting experiments on the four duplex DNAs ranged from -52.2 to -77.5 kcal/mol. Free energies based on the van't Hoff analysis were -37.9 to -58.8 kcal/mol. Although the values are different, trends in the melting free energies of the four duplex DNAs as a function of sequence were identical in both DSC and optical analyses. Subject to several assumptions, values for the initiation free energy were estimated for each duplex, defined as DeltaG(int) = DeltaG(cal) - DeltaG(pred), where DeltaG(cal) is the experimental free energy at 20 degrees C determined from the experimentially measured values of the transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal). The predicted free energy of the sequence, DeltaG(pred)(20 degrees C), is obtained using published nearest-neighbor sequence stability values. For three of the four duplexes, values of DeltaG(int) are essentially nil. In contrast, the duplex with 81.8% GC has a considerably higher estimate of DeltaG(int) = 7.1 kcal/mol. The CD spectra for the six duplexes collected over the wavelength range from 200 to 320 nm are also sequence-dependent. Factor analysis of the CD spectra by singular value decomposition revealed that the experimental CD spectra could be reconstructed from linear combinations of two minor and one major subspectra. Changes in the coefficients of the major subspectrum for different sequences reflect incremental sequence-dependent variations of the CD spectra. Equilibrium binding by BamHI restriction endonuclease to the 40 base pair DNAs whose central eight base pairs contain the recognition sequence for BamHI restriction enzyme bounded by A.T base pairs, 5'-A-GGATCC-A-3' was investigated. Binding assays were performed by titering BamHI against a constant concentration of each of the duplex DNA substrates, in the absence of Mg(2+), followed by analysis by gel retardation. Under the conditions employed, the enzyme binds but does not cleave the DNAs. Results of the assays revealed two binding modes with retarded gel mobilities. Binding isotherms for the fraction of bound DNA species versus enzyme concentration for each binding mode were constructed and analyzed with a simple two-step equilibrium binding model. This analysis provided semiquantitative estimates on the equilibrium binding constants for BamHI to the four DNAs. Values obtained for the binding constants varied only 7-fold and ranged from 6 x 10(-)(8) to 42 x 10(-)(8) M, with binding free energies from -8.6 to -9.7 (+/- 0.2) kcal/mol depending on the sequence that flanks the enzyme binding site. Unlike what was found earlier in binding studies of the 22 base pair duplexes that constitute the core modules of the present 40-mers [Riccelli, P. V., Vallone, P. M., Kashin, I., Faldasz, B. D., Lane, M. J., and Benight, A. S. (1999) Biochemistry 38, 11197-11208], no obvious relationship between binding and stability was found for these longer DNAs. Apparently, effects of flanking sequence stability on restriction enzyme binding may only be measurable in very short duplex deoxyoligonucl     

Is it always possible to distinguish two- and three-state systems by evaluating the van't Hoff enthalpy?

Many small globular proteins are traditionally classified as thermodynamical two-state systems, i.e., the protein is either in the native, active state (folded) or in the denatured state (unfolded). We challenge this view and show that there may exist (protein) systems for which a van't Hoff analysis of experimental data cannot determine whether the system corresponds to two or three thermodynamical states when only temperatures in a narrow temperature region around the transition are considered. We generalize a widely employed two-state protein folding model to include a third, transition state. For this three-state system we systematically study the deviation of the calorimetric enthalpy (heat of transition) from the van't Hoff enthalpy, a measure of the two-stateness of a transition. We show that under certain conditions the heat capacity of the three-state system can be almost indistinguishable from the heat capacity for the two-state system over a broad temperature interval. The consequence may be that some three-state (or even more than three-states) systems have been misinterpreted as two-state systems when the conclusion is drawn solely upon the van't Hoff enthalpy. These findings are important not only for proteins, but also for the interpretation of thermodynamical systems in general.     

Searching for quantitative entropy-enthalpy compensation among protein variants

Entropy-enthalpy (SH) compensation occurs when a small change in DeltaG is caused by large, and nearly compensatory, changes in DeltaH and DeltaS. It is considered a ubiquitous property of reactions in water. Because water is intimately involved in protein stability, SH compensation among protein variants, if it exists, could lead to important knowledge about protein-water interactions. In light of recent theoretical work on SH compensation, we gathered thermodynamic data for >200 protein variants to seek evidence for the simplest quantitative model of SH compensation (i.e., The van't Hoff denaturation enthalpy divided by the van't Hoff denaturation entropy is a constant). We conclude that either the data are insufficient to support the idea that quantitative SH compensation is a general feature of variant proteins or that such compensation does not exist. This study reinforces the idea that DeltaH-versus-DeltaS plots should not be used to provide evidence for SH compensation.     

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.