Thermodynamics of alligator metmyoglobin unfolding

The conformational free energy of alligator metmyoglobin was examined over a pH range of 4.4-8.0, a temperature range of 18-48 degrees C, and a guanidinium chloride concentration of 0-2.3 M. For isothermal unfolding at 25 degrees C essentially the same value was obtained for the conformational free energy from all the data: 7.0 +/- 0.5 kcal/mol. The cooperativity of the unfolding with respect to denaturant is considerably less than for mammalian myoglobins. On unfolding three to four side chains with a pKa of 6.3 in the unfolded protein are protonated instead of the six expected. The temperature at which delta H (unfolding) is zero is much lower than for previously characterized myoglobins. Alligator metmyoglobin, considerably less stable than other previously characterized myoglobins, may not be as compactly folded.     

Thermodynamics of carp metmyoglobin unfolding

The conformational free energy of carp lateral muscle metmyoglobin at 25 degrees C pH 8 is 9.0 +/- 0.5 kcal/mol as estimated from isothermal unfolding by both urea and guanidinium chloride. A novel procedure for the simultaneous analysis of acid and guanidinium chloride unfolding data is described. Acid denaturation data suggest that binding of five protons to histidyl residues occurs on unfolding. Correlation of sequences and conformational stabilities of several myoglobins according to the tertiary structure of sperm whale myoglobin suggests an evolutionary turnover of side chain-side chain interactions.     

pH-dependent stability of sperm whale myoglobin in water-guanidine hydrochloride solutions

An experimental-theoretical approach for the elucidation of protein stability is proposed. The theoretical prediction of pH-dependent protein stability is based on the macroscopic electrostatic model for calculation of the pH-dependent electrostatic free energy of proteins. As a test of the method we have considered the pH-dependent stability of sperm whale metmyoglobin. Two theoretical methods for evaluation of the electrostatic free energy and p K values are applied: the finite-difference Poisson-Boltzmann method and the semiempirical approach based on the modified Tanford-Kirkwood theory. The theoretical results for electrostatic free energy of unfolding are compared with the experimental data for guanidine hydrochloride unfolding under equilibrium conditions over a wide pH range. Using the optical parameters of the Soret absorbance to monitor conformational equilibrium and Tanford's method to estimate the resulting data, it was found that the conformational free energy of unfolding of metmyoglobin is 16.3 kcal mol(-1) at neutral pH values. The total unfolding free energies were calculated on the basis of the theoretically predicted electrostatic unfolding free energies and the experimentally measured midpoints (pH(1/2)) of acidic and alkaline denaturation transitions. Experimental data for alkaline denaturation were used for the first time in theoretical analysis of the pH-dependent unfolding of myoglobin. The present results demonstrate that the simultaneous application of appropriate theoretical and experimental methods permits a more complete analysis of the pH-dependent and pH-independent properties and stability of globular proteins.     

A comparative study of the unfolding thermodynamics of vertebrate metmyoglobins

Differential scanning microcalorimetry (DSC) of horse, rat, opossum, raccoon, carp, and armadillo metmyoglobins at alkaline pH gave data that fit the two-state unfolding model well. Monte Carlo studies were used to assess the impact of truncating DSC scans on the reliability of the calculated results when aggregation exotherms overlapped the unfolding endotherm at the high-temperature end of the scan. The DSC estimates for the conformational free energy at pH 8 and 298 K are compared to earlier results from isothermal acid and guanidinium chloride unfolding. Stability estimates at pH 8 for these six metmyoglobins obtained by DSC experiments do not agree with free energy estimates at pH 8 from guanidinium chloride unfolding. This is true for all three models used to extrapolate the free energy change to 0 M guanidinium chloride. Among these six myoglobins, significant variation appears in the temperature at which the myoglobin is half-unfolded, in the change in heat capacity upon unfolding, and in the change in enthalpy at 310 K. Calculations made with the hydrophobic model for protein folding [Baldwin, R.L. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8069] suggest that a sizable variation exists for that portion of the unfolding enthalpy change assigned to forces other than the hydrophobic effect.     

Denaturation thermodynamics of chicken cardiac metmyoglobin

The unfolding at pH 8 of chicken cardiac aquometmyoglobin was examined as a function of temperature and concentration of guanidinium chloride using the two-state model. The isothermal unfolding data at 25°C were fitted to Tanford's transfer model and the binding model of Aune and Tanford. The estimates obtained for ΔG_D) were virtually identical, viz., 8.3 ±0.3 kcal mol^?1. The chicken metmyoglobin is thus some 5.3 kcal mol^?1 less stable than that of sperm whale metmyoglobin. The unfolding parameters α and Δn were decreased 20% from those of mammalian myoglobins thus far examined, suggesting nonidentity of native conformations. The apparent enthalpy change on unfolding was dependent on both temperature and denaturant concentration. The decreases in the isothermal unfolding parameters from those of sperm whale are principally assigned to three of the 46 sequence changes.     

Conformational free energies of myoglobins of small mammals

Myoglobins from three small placental mammals and one small marsupial were isolated from cardiac or skeletal muscle. The conformational free energies of these four myoglobins were estimated from guanidinium chloride unfolding data at pH 8 and 25 degrees. The myoglobins from rat and rabbit are more stable than that of the most stable myoglobin previously studied, that of the sperm whale. In addition, these two myoglobins unfold with greater cooperativity than previously characterized myoglobins. The data obtained herein demonstrate unequivocally for the first time that the stability of homeotherm myoglobins correlates with neither the size of the organism nor its metabolic rate.     

Dimeric procaspase-3 unfolds via a four-state equilibrium process.

We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).     

Thermodynamic analysis of the low- to physiological-temperature nondenaturational conformational change of bovine carbonic anhydrase

The stability curve - a plot of the Gibbs free energy of unfolding versus temperature - is calculated for bovine erythrocyte carbonic anhydrase in 150 mM sodium phosphate (pH = 7.0) from a combination of reversible differential scanning calorimetry measurements and isothermal guanidine hydrochloride titrations. The enzyme possesses two stable folded conformers with the conformational transition occurring at ~30 degrees C. The methodology yields a stability curve for the complete unfolding of the enzyme below this temperature but only the partial unfolding, to the molten globule state, above it. The transition state thermodynamics for the low- to physiological-temperature conformational change are calculated from slow-scan-rate differential scanning calorimetry measurements where it is found that the free energy barrier for the conversion is 90 kJ/mole and the transition state possesses a substantial unfolding quality. The data therefore suggest that the x-ray structure may differ considerably from the physiological structure and that the two conformers are not readily interconverted.     

Effect of chaotropic agents on reversible unfolding of a soybean (Glycine max) seed acid phosphatase

In this work we examined the effect of urea and guanidinium chloride on the structural stability of a single isoform of soybean seed acid phosphatase, based on the intensity of tryptophan fluorescence as a function of denaturant concentration. The free energy of unfolding, DeltaGu, was calculated at 25 degrees C as a function of the concentrations of both chaotropic agents; the conformational stability, DeltaG (H2O), was determined to be 2.48 kcal mol(-1). Center of mass, determined from analysis of fluorescence data, was used as a parameter to assess conformational changes. Our results indicate that complete enzyme inactivation occurred before full enzyme unfolding in both cases, and suggest that there are differences between the conformational flexibility of the active-site and that of the macromolecule as a whole.     

The conformational stability and flexibility of insulin with an additional intramolecular cross-link

The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.     

Comparison between denaturant- and temperature-induced unfolding pathways of protein: a lattice Monte Carlo simulation

Denaturant-induced unfolding of protein is simulated by using a Monte Carlo simulation with a lattice model for protein and denaturant. Following the binding theory for denaturant-induced unfolding, the denaturant molecules are modeled to interact with protein by nearest-neighbor interactions. By analyzing the conformational states on the unfolding pathway of protein, the denaturant-induced unfolding pathway is compared with the temperature-induced unfolding pathway under the same condition; that is, the free energies of unfolding under two different pathways are equal. The two unfoldings show markedly different conformational distributions in unfolded states. From the calculation of the free energy of protein as a function of the number fraction (Q0) of native contacts relative to the total number of contacts, it is found that the free energy of the largely unfolded state corresponding to low Q0 (0.1 < Q0 < 0.5) under temperature-induced unfolding is lower than that under denaturant-induced unfolding, whereas the free energy of the unfolded state close to the native state (Q0 > 0.5) is lower in denaturant-induced unfolding than in temperature-induced unfolding. A comparison of two unfolding pathways reveals that the denaturant-induced unfolding shows a wider conformational distribution than the temperature-induced unfolding, while the temperature-induced unfolding shows a more compact unfolded state than the denaturant-induced unfolding especially in the low Q0 region (0.1 < Q0 < 0.5).     

Comparative studies of unfolding and binding of ligands to human serum albumin in the presence of fatty acid: spectroscopic approach

This study was undertaken to investigate the influence of fatty acid binding on the unfolding of HSA and how the fatty acid molecules can influence and/or compete with other ligand molecules bound to the protein. The equilibrium unfolding of fatted and fatty acid free HSA was measured by overlapping of unfolding transition curves monitored by different probes for secondary and tertiary structure and determining changes in free energy of unfolding. Proteins stability was studied by fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism techniques. We have suggested a "molten globule" like intermediate state of HSA at a fairly high concentration of GnHCl (3.2 for fatty acid free and 3.6 for fatted). The free energy of stabilization (DeltaG(D)(H2O)) in the presence of fatty acid was found to be 900 cal mol(-1). We also analyze the effects of fatty acid on binding of ligands using spectroscopic technique and reported the equilibrium constants and free energies obtained from the binding and unfolding experiments.     

High temperature unfolding of a truncated hemoglobin by molecular dynamics simulation

Heme containing proteins are associated with peroxidase activity. The proteins like hemoglobin, myoglobins, cytochrome c and micro-peroxidase other than peroxidases have been shown to exhibit weak peroxidase-like activity. This weak peroxidase–like activity in hemoglobin-like molecules is due to heme moiety. We conducted molecular dynamics (MD) studies to decipher the unfolding path of Ba-Glb (a truncated hemoglobin from Bacillus anthracis) and the role of heme moiety to its unfolding path. The similar unfolding path is also observed in vitro by UV/VIS spectroscopy. The data confirmed that the unfolding of Ba-Glb follows a three state process with a meta-stable (intermediate) state between the native and unfolded conformations. The present study is supported by several unfolding parameters like root-mean-square-deviation (RMSD), dictionary of protein secondary structure (DSSP), and free energy landscape. Understanding the structure of hemoglobin like proteins in unicellular dreaded pathogens like B. anthracis will pave way for newer drug discovery targets and in the disease management of anthrax.     

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.     

Thermodynamics of globular proteins

The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the ‘gas phase’. It was shown that specific values of the energy of weak intramolecular bonds (?H^int), conformational free energy (?G^conf) and entropy (?S^conf) are the same for proteins with molecular weight 7–25 kDa. Using the energy value (?H^int) and the proposed approach for estimation of the conformational entropy of native protein (S^NC), numerical values of the absolute free energy (G^NC) were obtained.     

Axial ligand effects on myoglobin stability

Reversible guanidine hydrochloride denaturation has been applied to obtain the first quantitative estimate of ligand-induced changes in hemoprotein conformational free energy. It is found that strong field (low spin) complexes, e.g. cyanometmyoglobin (MbCN) and azido metmyoglobin (MbN3), are 1.0 +/- 0.1 kcal/mol more stable than the high spin analogs aquometmyoglobin (MbH2O) and fluorometmyoglobin (MbF). This observed stability increment is essentially independent of the model chosen for data analysis. These results demonstrate the value of denaturation titration in measuring the stabilization of hemoprotein conformation by ligand binding. The denaturation of MbN3 appears complex. This complexity may be quantitatively accounted for by considering spin state equilibria. Applying this correction, MbCN and MbN3 have essentially the same stability in spite of steric differences in the two proteins. This result implies metal spin state is more important than ligand stereochemistry in determining the conformational free energy of myoglobin.     

The entropic nature of protein thermal stabilization

We performed thermodynamic analysis of temperature-induced unfolding of mesophilic and thermophilic proteins. It was shown that the variability in protein thermostability associated with pH-dependent unfolding or linked to the substitution of amino acid residues on the protein surface is evidence of the governing role of the entropy factor. Numerical values of conformational components in enthalpy, entropy and free energy which characterize protein unfolding in the “gas phase” were obtained. Based on the calculated absolute values of entropy and free energy, a model of protein unfolding is proposed in which the driving force is the conformational entropy of native protein, as an energy of the heat motion (T·S^NC) increasing with temperature and acting as an factor devaluating the energy of intramolecular weak bonds in the transition state.     

Thermodynamic analysis of the nondenaturational conformational change of baker's yeast phosphoglycerate kinase at 24 degrees C

A plot of the Gibbs free energy of unfolding vs. temperature is calculated for baker’s yeast phosphoglycerate kinase in 150 mM sodium phosphate (pH = 7.0) from a combination of reversible differential scanning calorimetry measurements and isothermal guanidine hydrochloride titrations. The stability curve reveals the existence of two stable, folded conformers with an abrupt conformational transition occurring at 24 °C. The transition state thermodynamics for the low- to high-temperature conformational change are calculated from slow-scan-rate differential scanning calorimetry measurements where it is found that the free energy barrier for the conversion is 90 kJ/mol and the transition state possesses a significant unfolding quality. This analysis also confirms a nondenaturational conformational transition at 24 °C. The data therefore suggest that X-ray structures obtained from crystals grown below this temperature may differ considerably from the physiological structure and that the two conformers are not readily interconverted.     

Amino acid composition and physico-chemical properties of bluefin tuna (Thunnus thynnus) myoglobin.

1. The heart ventricle myoglobin of Atlantic bluefin tuna has been purified and its amino acid composition has been determined. 2. The perturbing effect of guanidine hydrochloride on the molecular structure of tuna ferrimyoglobin and its corresponding apoprotein has been investigated by Soret absorbance and ultraviolet fluorescence. 3. The conformation-free energy of unfolding delta G0 has been calculated by thermodynamic treatments of the data concerning guanidine unfolding. 4. The results have been compared with other known myoglobins, particularly those of yellowfin tuna.     

Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.