Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.     

Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (tm) were 43.9 +/- 1.4 and 63.3 +/- 1.6 degrees C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +/- 1.3 degrees C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase.     

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.     

Calorimetric and spectroscopic investigation of the unfolding of human apolipoprotein B

The unfolding of human apolipoprotein B-100 in its native lipid environment, low density lipoprotein (LDL), and in a soluble, lipid-free complex with sodium deoxycholate (NaDC) has been examined using differential scanning calorimetry (DSC) and near UV circular dichroic (CD) spectroscopy. High resolution DSC shows that LDL undergoes three thermal transitions. The first is reversible and corresponds to the order-disorder transition of the core-located cholesteryl esters (CE) (Tm = 31.1 degrees C, delta H = 0.75 cal/g CE). The second, previously unreported, is reversible with heating up to 65 degrees C (Tm = 57.1 degrees C, delta H = 0.20 cal/g apoB) and coincides with a reversible change in the tertiary structure of apoB as shown by near UV-CD. No alteration in the secondary structure of apoB is observed over this temperature range. The third transition is irreversible (Tm = 73.5 degrees C, delta H = 0.99 cal/g apoB) and coincides with disruption of the LDL particle and denaturation of apoB. The ratio of delta H/delta HvH for the reversible protein-related transition suggests that this is a two-state event that correlates with a change in the overall tertiary structure of the entire apoB molecule. The second protein-related transition is complex and coincides with irreversible denaturation. ApoB solubilized in NaDC undergoes three thermal transitions. The first two are reversible (Tm = 49.7 degrees C, delta H = 1.13 cal/g apoB; Tm = 56.4 degrees C, delta H = 2.55 cal/g apoB, respectively) and coincide with alterations in both secondary and tertiary structure of apoB. The changes in secondary structure reflect an increase in random coil conformation with a concomitant decrease in beta-structure, while the change in tertiary structure suggests that the conformation of the disulfide bonds is altered. The third transition is irreversible (Tm = 66.6 degrees C, delta H = 0.54 cal/g apoB) and coincides with complete denaturation of apoB and disruption of the NaDC micelle. The ratio of delta H/delta HvH for the two reversible transitions indicates that each of these transitions is complex which may suggest that several regions or domains of apoB are involved in each thermal event.     

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T_m ≈ 100 °C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry–Eyring model, N ? U ? F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.     

Calorimetric study of the heat and cold denaturation of beta-lactoglobulin.

Temperature-induced changes of the states of beta-lactoglobulin have been studied calorimetrically. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. Its heat denaturation is approximated very closely by a two-state transition, while the cold denaturation deviates considerably from the two-state transition and this deviation increases as the temperature decreases. The heat effect of cold denaturation is opposite in sign to that of heat denaturation and is noticeably larger in magnitude. This difference in magnitude is caused by the temperature-dependent negative heat effect of additional binding of urea to the polypeptide chain of the protein upon its unfolding, which decreases the positive enthalpy of heat denaturation and increases the negative enthalpy of cold denaturation. The binding of urea considerably increases the partial heat capacity of the protein, especially in the denatured state. However, when corrected for the heat capacity effect of urea binding, the partial heat capacity of the denatured protein is close in magnitude to that expected for the unfolded polypeptide chain in aqueous solution without urea but only for temperatures below 10 degrees C. At higher temperatures, the heat capacity of the denatured protein is lower than that expected for the unfolded polypeptide chain. It appears that at temperatures above 10 degrees C not all the surface of the beta-lactoglobulin polypeptide chain is exposed to the solvent, even in the presence of 6 M urea; i.e., the denatured protein is not completely unfolded and unfolds only at temperatures lower than 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolar and polar solvation thermodynamics related to the protein unfolding process.

Thermodynamics related to hydrated water upon protein unfolding is studied over a broad temperature range (5-125 degrees C). The hydration effect arising from the apolar interior is modeled as an increased number of hydrogen bonds between water molecules compared with bulk water. The corresponding contribution from the polar interior is modeled as a two-step process. First, the polar interior breaks hydrogen bonds in bulk water upon unfolding. Second, due to strong bonds between the polar surface and the nearest water molecules, we assume quantization using a simplified two-state picture. The heat capacity change upon hydration is compared with model compound data evaluated previously for 20 different proteins. We obtain good correspondence with the data for both the apolar and the polar interior. We note that the effective coupling constants for both models have small variations among the proteins we have investigated.     

Hydration and thermal denaturation of beta-lactoglobulin. A calorimetric study.

The thermal properties of the beta-lactoglobulin-water system were investigated by differential scanning calorimetry in the temperature range from -50 to 130 degrees C. Determination of the heat and temperature of fusion of the absorbed water allowed resolution of the water into four different states. The amounts of water in these states were different for samples before and after heat denaturation. In the case of denatured beta-lactoglobulin, a smaller amount of water with thermal properties different from ordinary water was observed and its total water binding capacity was lower. The thermal stability of beta-lactoglobulin in the water content range from 0 to 0.75 g/g showed a strong dependence on the degree of hydration. A correlation was observed between the changes in the thermal stability of the protein and the changes in the state of the absorbed water. The results are compared with those obtained from similar measurements of other globular proteins and of fibrillar proteins.     

Solid model compounds and the thermodynamics of protein unfolding.

Analysis of thermodynamic data on the dissolution of solid cyclic dipeptides into water in terms of group additivity provides a rationale for the enthalpy and entropy convergence temperatures observed for small globular protein denaturation and the dissolution of model compounds into water. Convergence temperatures are temperatures at which the extrapolated enthalpy or entropy changes for a series of related compounds take on a common value. At these temperatures (TH* and TS*) the apolar contributions to the corresponding thermodynamic values (delta H degrees and delta S degrees) are shown to be zero. Other contributions such as hydrogen bonding and configurational effects can then be evaluated and their quantitative effects on the stability of globular proteins assessed. It is shown that the denaturational heat capacity is composed of a large positive contribution from the exposure of apolar groups and a significant negative contribution from the exposure of polar groups in agreement with previous results. The large apolar contribution suggests that a liquid hydrocarbon model of the hydrophobic effect does not accurately represent the apolar contribution to delta H degrees of denaturation. Rather, significant enthalpic stabilizing contributions are found to arise from peptide groups (hydrogen bonding). Combining the average structural features of globular proteins (i.e. number of residues, fraction of buried apolar groups and fraction of hydrogen bonds) with their specific group contributions permits a first-order prediction of the thermodynamic properties of proteins. The predicted values compare well with literature values for cytochrome c, myoglobin, ribonuclease A and lysozyme. The major thermodynamic features are described by the number of peptide and apolar groups in a given protein.     

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.     

Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation

The conformational stability of RNase Rs was determined with chemical and thermal denaturants over the pH range of 1-10. Equilibrium unfolding with urea showed that values of D(1/2) (5.7 M) and DeltaG(H(2)O) (12.8 kcal/mol) were highest at pH 5.0, its pI and the maximum conformational stability of RNase Rs was observed near pH 5.0. Denaturation with guanidine hydrochloride (GdnHCl), at pH 5.0, gave similar values of DeltaG(H(2)O) although GdnHCl was 2-fold more potent denaturant with D(1/2) value of 3.1 M. The curves of fraction unfolded (f(U)) obtained with fluorescence and CD measurements overlapped at pH 5.0. Denaturation of RNase Rs with urea in the pH range studied was reversible but the enzyme denatured irreversibly >pH 11.0. Thermal denaturation of RNase Rs was reversible in the pH range of 2.0-3.0 and 6.0-9.0. Thermal denaturation in the pH range 4.0-5.5 resulted in aggregation and precipitation of the protein above 55 degrees C. The aggregate was amorphous or disordered precipitate as observed in TE micrographs. Blue shift in emission lambda(max) and enhancement of fluorescence intensity of ANS at 70 degrees C indicated the presence of solvent exposed hydrophobic surfaces as a result of heat treatment. Aggregation could be prevented partially with alpha-cyclodextrin (0.15 M) and completely with urea at concentrations >3 M. Aggregation was probably due to intermolecular hydrophobic interaction favored by minimum charge-charge repulsion at the pI of the enzyme. Both urea and temperature-induced denaturation studies showed that RNase Rs unfolds through a two-state F right arrow over left arrow U mechanism. The pH dependence of stability described by DeltaG(H(2)O) (urea) and DeltaG (25 degrees C) suggested that electrostatic interactions among the charged groups make a significant contribution to the conformational stability of RNase Rs. Since RNase Rs is a disulfide-containing protein, the major element for structural stability are the covalent disulfide bonds.     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.     

Two-phase unfolding pathway of ribonuclease A during denaturation induced by dithiothreitol

The dynamics of the unfolding process of bovine pancreatic ribonuclease A (RNase A) unfolded by dithiothreitol (DTT) at a low concentration of 1:30 were investigated in alkaline phosphate-buffered saline solutions at 303K and 313K by using proton nuclear magnetic resonance ((1)H NMR) spectra. Three NMR spectral parameters including Shannon entropy, mutual information, and correlation coefficient were introduced into the analysis. The results show that the unfolding process of RNase A was slowed to the order of many hours, and the kinetics of the unfolding pathway described by the three parameters is best fit by a model of two consecutive first-order reactions. Temperature greatly influences the rate constants of the unfolding kinetics with different temperature effects observed for the fast and the slow processes. The consistencies and the differences between the three sets of parameters show that they reflect the same relative denaturation pathway but different spectra windows of the unfolding process of RNase A. The results suggest that the unfolding process of RNase A induced by low concentrations of DTT is a two-phase pathway containing fast and slow first-order reactions.     

Calorimetric dissection of thermal unfolding of OspA,a predominantly beta-sheet protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from Borrelia burgdorferi is a predominantly beta-sheet protein comprised of beta-strands beta1-beta21 and a short C-terminal alpha-helix. It contains two globular domains (N and C-terminal domains) and a unique single-layer beta-sheet (central beta-sheet) that connects the two domains. OspA contains an unusually large number of charged amino acid residues. To understand the mechanism of stabilization of this unique beta-sheet protein, thorough thermodynamic investigations of OspA and its truncated mutant lacking a part of the C-terminal domain were conducted using calorimetry and circular dichroism. The stability of OspA was found to be sensitive to pH and salt concentration. The heat capacity curve clearly consisted of two components, and all the thermodynamic parameters were obtained for each step. The thermodynamic parameters associated with the two transitions are consistent with a previously proposed model, in which the first transition corresponds to the unfolding of the C-terminal domain and the last two beta-strands of the central beta-sheet, and the second transition corresponds to that of the N-terminal domain and the first beta-strand of the central beta-sheet in the second peak. The ratio of calorimetric and van't Hoff enthalpies indicates that the first peak includes another thermodynamic intermediate state. Large heat capacity changes were observed for both transitions, indicative of large changes in the exposure of hydrophobic surfaces associated with the transitions. This observation demonstrates that hydrophobic parts are buried efficiently in the native structure in spite of the low content of hydrophobic residues in OspA. By decomposing the enthalpy, entropy, and Gibbs free energy into contributions from different interactions, we found that the enthalpy changes for hydrogen bonding and polar interactions are exceptionally large, indicating that OspA maintains its stability by making full use of its unique beta-sheet and high content of polar residues. These thermodynamic analyses demonstrated that it is possible to maintain protein tertiary structure by making effective use of an unusual amino acid composition.     

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.     

Biphasic denaturation of human albumin due to ligand redistribution during unfolding

Denaturation of defatted human albumin monomer, monitored by differential scanning calorimetry, is monophasic as reflected by the single, resulting endotherm. With low levels of various ligands, biphasic or monophasic unfolding processes are manifested as bimodal or unimodal thermograms, respectively. The greater the affinity of native protein for ligand, the greater is the tendency for biphasic denaturation. We propose that such a biphasic unfolding process arises from a substantial increase in stability (transition temperature) of remaining native protein during denaturation. This increase in stability derives from the free energy of ligand binding becoming more negative due to the release of high affinity ligand by unfolding protein. The tendency for biphasic denaturation is greatest at low (subsaturating) levels of ligand where greatest increases in stability occur. Biphasic unfolding arising from such ligand redistribution results from denaturation of different kinds of protein molecules, ligand-poor and ligand-rich species, and not from sequential unfolding of domains within the same molecule. Differentiating between these two mechanisms is necessary for the correct interpretation of biphasic denaturation data. Furthermore, biphasic unfolding due to ligand redistribution occurs independently of the means used to effect denaturation. The maximum increase in stability due to ligand binding relative to the stability of defatted albumin monomer alone occurs with the intermediate affinity ligand octanoate (22 degrees C) and not with the high affinity ligand hexadecanoate (15 degrees C). This indicates a much greater affinity of denatured albumin for hexadecanoate since increase in stability derives from the difference between free energy of ligand binding to folded and unfolded protein forms.