Two-state Irreversible Model Research Articles

A strategy based on thermal flexibility to design triosephosphate isomerase proteins with increased or decreased kinetic stability

Kinetic stability of proteins determines their susceptibility to irreversibly unfold in a time-dependent process, and therefore its half-life. A residue displacement analysis of temperature-induced unfolding molecular dynamics simulations was recently employed to define the thermal flexibility of proteins. This property was found to be correlated with the activation energy barrier (Eact) separating the native from the transition state in the denaturation process. The Eact was determined from the application of a two-state irreversible model to temperature unfolding experiments using differential scanning calorimetry (DSC). The contribution of each residue to the thermal flexibility of proteins is used here to propose multiple mutations in triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), two parasites closely related by evolution. These two enzymes, taken as model systems, have practically identical structure but large differences in their kinetic stability. We constructed two functional TIM variants with more than twice and less than half the activation energy of their respective wild-type reference structures. The results show that the proposed strategy is able to identify the crucial residues for the kinetic stability in these enzymes. As it occurs with other protein properties reflecting their complex behavior, kinetic stability appears to be the consequence of an extensive network of inter-residue interactions, acting in a concerted manner. The proposed strategy to design variants can be used with other proteins, to increase or decrease their functional half-life.

Irreversible thermal denaturation of Helix aspersa maxima hemocyanin

The thermal unfolding of hemocyanin, purified from garden snails Helix aspersa maxima (gastropod) (HaH), was investigated by differential scanning calorimetry (DSC), far-UV circular dichroism (CD) and UV–Vis spectroscopy. Denaturation of HaH was found to be an irreversible process. One transition, with an apparent transition temperature (Tm) at 79.8 °C, was detected by DSC in the thermogram of HaH in 20 mM HEPES buffer, containing 0.1 M NaCl, 5 mM CaCl2 and 5 mM MgCl2, pH 7.0, using scan rate of 1.0 °C min−1. The scan rate dependence of the calorimetric profiles indicated that the thermal unfolding of investigated HaH was kinetically controlled. The Tm and calorimetric enthalpy values (ΔHcal) for the thermal denaturation of HaH were found to be independent of the protein concentration, indicating that the dissociation of the hemocyanin into subunits does not take place before the rate-determining step of the process of thermal unfolding started. The calorimetrically observed thermal transition showed excellent coincidence with the unfolding transition monitored by CD measurements. The thermal denaturation of HaH was described by the two-state irreversible model, and parameters of the Arrhenius equation were calculated.

Calorimetric Study of Helix aspersa Maxima Hemocyanin Isoforms.

The thermal unfolding of hemocyanin isoforms, β-HaH and αD+N-HaH, isolated from the hemolymph of garden snails Helix aspersa maxima, was studied by means of differential scanning calorimetry (DSC). One transition, with an apparent transition temperature (Tm) at 79.88°C, was detected in the thermogram of β-HaH in 20 mM HEPES buffer, containing 0.1 M NaCl, 5 mM CaCl2, and 5 mM MgCl2, pH 7.0, at scan rate of 1.0°C min−1. By means of successive annealing procedure, two individual transitions were identified in the thermogram of αD+N-HaH. Denaturation of both hemocyanins was found to be an irreversible process. The scan-rate dependence of the calorimetric profiles indicated that the thermal unfolding of investigated hemocyanins was kinetically controlled. The thermal denaturation of the isoforms β-HaH and αD+N-HaH was described by the two-state irreversible model, and parameters of the Arrhenius equation were calculated.

Osmolytic Effect of Sucrose on Thermal Denaturation of Pea Seedling Copper Amine Oxidase.

Protein stability is a subject of interest by many researchers. One of the common methods to increase the protein stability is using the osmolytes. Many studies and theories analyzed and explained osmolytic effect by equilibrium thermodynamic while most proteins undergo an irreversible denaturation. In current study we investigated the effect of sucrose as an osmolyte on the thermal denaturation of pea seedlings amine oxidase by the enzyme activity, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry. All experiments are in agreement that pea seedlings amine oxidase denaturation is controlled kinetically and its kinetic stability is increased in presence of sucrose. Differential scanning calorimetry experiments at different scanning rates showed that pea seedlings amine oxidase unfolding obeys two-state irreversible model. Fitting the differential scanning calorimetry data to two-state irreversible model showed that unfolding enthalpy and T *, temperature at which rate constant equals unit per minute, are increased while activation energy is not affected by increase in sucrose concentration. We concluded that osmolytes decrease the molecular oscillation of irreversible proteins which leads to decline in unfolding rate constant.

The influence of active site loop mutations on the thermal stability of azurin from Pseudomonas aeruginosa

The copper site and overall structures of azurin (AZ) variants in which the amicyanin (AMI) and plastocyanin (PC) metal binding loops have been introduced, AZAMI and AZPC, respectively, are similar to that of AZ, whereas the loop conformations resemble those in the native proteins. To assess the influence of these loop mutations on stability, the thermal unfolding of AZAMI and AZPC has been investigated by differential scanning calorimetry, absorption and fluorescence spectroscopy. The calorimetric profiles of both variants exhibit a complex shape consisting of two endothermic peaks and an exothermic peak. The temperature of the maximum heat of absorption for the single endothermic peak is 82.7°C for AZ, whereas for AZAMI and AZPC the most intense endothermic peaks are at 74.9 and 68.1°C comparable to values for AMI and PC, respectively. Denaturation investigated using the temperature dependence of the absorbance at ∼600nm and Trp emission, also demonstrates decreased stability for both loop mutants. The thermal transition between the native and the denaturated states is irreversible, scan rate dependent and consistent with the two-state irreversible model. The structure of the active-site loop has a dramatic effect on the kinetic stability and the unfolding pathway of cupredoxins.

The Bilayer Enhances Rhodopsin Kinetic Stability in Bovine Rod Outer Segment Disk Membranes

Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl- β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl- β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature ( T m) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the T m as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation ( E act) was calculated from the scan rate dependence of the T m and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The E act as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the E act to rhodopsin denaturation.

Spectroscopic Properties and Conformational Stability of Concholepas concholepas Hemocyanin

The structure in solution and conformational stability of the hemocyanin from the Chilean gastropod mollusk Concholepas concholepas (CCH) and its structural subunits, CCH-A and CCH-B, were studied using fluorescence spectroscopy and differential scanning calorimetry (DSC). The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Besides tryptophan residues buried in the hydrophobic interior of the protein molecule also exposed fluorophores determine the fluorescence emission of the oxy- and apo-forms of the investigated hemocyanins. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-forms of CCH and its subunits. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophan residues in the respective apo-forms. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the CCH and its subunits exist in different conformations. The thermal denaturation of the hemocyanin is an irreversible process, under kinetic control. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Arrhenius equation parameter for the two-state irreversible model of the thermal denaturation of oxy-CCH at pH 7.2 was estimated. Both factors, oligomerization and the copper-dioxygen system at the active site, are important for stabilizing the structure of the hemocyanin molecule.

Calorimetric studies on renaturation by CaCl2 addition of metal-free α-amylase from Bacillus Licheniformis (BLA)

In our previous work we characterized the Ca binding properties of BLA by analysis of Ca-induced renaturation in the presence of urea. In this study we focused on the renaturing capacity of CaCl2 in the absence of urea and analysed the apparent thermodynamic and kinetic properties of renatured BLA by DSC, CD and dynamic light scattering (DLS) measurements. Ca-free protein did not return fully to the native state even after extensive dialysis against high concentrations of calcium. Thus Ca removal provokes changes in protein structure that can not be reversed completely even by addition of an excess of calcium. However, activity studies performed simultaneously with the DSC experiments showed a significant return of enzymatic activity despite the failure of complete return of native stability. This conclusion is in excellent agreement with findings of Machius et al. who suggested on the basis of their X-ray studies that it is not possible to remove CaI and CaII without introducing significant structural changes. It is important to note that all unfolding reactions of metal-free, partially renatured and native protein were found to be irreversible. Therefore the DSC transition curves were fitted using irreversible transition models. It turned out that the apparent heat capacity profiles of partially renatured BLA could be well represented by superposition of two irreversible processes each following the two-state irreversible model.

Irreversible Thermal Denaturation of β-Hemocyanin of Helix pomatia and its Substructures Studied by Differential Scanning Calorimetry

The thermal denaturation of β -hemocyanin from the gastropod Helix pomatia (β -HpH) at neutral pH was studied by means of differential scanning calorimetry (DSC). The denaturation was completely irreversible as judged by the absence of any endotherm on rescanning previously scanned samples. Two transitions, with apparent transition temperatures (Tm) of ca. 84 °C (main transition) and ca. 88 °C (minor transition), were detected by DSC in 20 mM MOPS buffer, containing 0.1 M NaCl, 5mM CaCl2 and 5 mM MgCl2 at pH 7.2 (buffer A), using a heating rate of 1.0 Kmin−1. Both Tm values were dependent on the scanning rate, suggesting that the thermal denaturation of β -HpH is a kinetically controlled process. The Tm and specific enthalpy values (ΔHcal) for the thermal denaturation of β -HpH were found to be independent of the protein concentration, indicating that the dissociation of the protein into monomers does not take place before the rate-determining step of the process of thermal unfolding started. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. These transitions are tentatively attributed to the denaturation of, respectively, the wall (main transition) and the collar of the β -HpH molecule. The activation energies (EA) of both transitions were found to be similar (about 500 kJ mol−1). In 130 mM glycine/NaOH buffer, pH 9.6 (buffer B), with β -HpH dissociated into subunits, the calorimetric profile had a more complex character. This could be ascribed to a different stability of the functional units (FUs) constituting the β -HpH subunit. FU d, which in the cylindrical didecameric β -HpH molecule is located in the wall, was markedly less stable than FU g, which belongs to the collar. The thermal denaturation of FUs d and g was described by the two-state irreversible model. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Role of Solvation Barriers in Protein Kinetic Stability

The stability of several protein systems of interest has been shown to have a kinetic basis. Besides the obvious biotechnological implications, the general interest of understanding protein kinetic stability is emphasized by the fact that some emerging molecular approaches to the inhibition of amyloidogenesis focus on the increase of the kinetic stability of protein native states. Lipases are among the most important industrial enzymes. Here, we have studied the thermal denaturation of the wild-type form, four single-mutant variants and two highly stable, multiple-mutant variants of lipase from Thermomyces lanuginosa. In all cases, thermal denaturation was irreversible, kinetically controlled and conformed to the two-state irreversible model. This result supports that the novel molecular-dynamics-focused, directed-evolution approach involved in the preparation of the highly stable variants is successful likely because it addresses kinetic stability and, in particular, because heated molecular dynamics simulations possibly identify regions of disrupted native interactions in the transition state for irreversible denaturation. Furthermore, we find very large mutation effects on activation enthalpy and entropy, which were not accompanied by similarly large changes in kinetic urea m-value. From this we are led to conclude that these mutation effects are associated to some structural feature of the transition state for the irreversible denaturation process that is not linked to large changes in solvent accessibility. Recent computational studies have suggested the existence of solvation/desolvation barriers in at least some protein folding/unfolding processes. We thus propose that a solvation barrier (arising from the asynchrony between breaking of internal contacts and water penetration) may contribute to the kinetic stability of lipase from T. lanuginosa (and, possibly, to the kinetic stability of other proteins as well).

DSC study of glycerol-extracted muscle fibers in intermediate states of ATP hydrolysis

The heat capacity of contractile proteins actin and myosin was studied in psoas muscle of rabbit in strongly and weakly binding state of myosin to actin as a function of temperature by DSC. Deconvolution of the unfolding scans makes possible to characterize the structural domains of the macromolecules. We tried to approach the unfolding process in different intermediate state of ATP hydrolysis. The thermal transitions were calorimetrically irreversible, therefore the two-state irreversible model that describes fairly well the denaturation of different proteins was used for evaluation of the denaturation processes in muscle fibers in strongly (rigor, ADP) and weakly binding states (ATP·Vi, ADP·AlF4) of myosin to actin. Deconvolution resulted in four transitions, the first three transition temperatures were almost independent of the intermediate states of muscle, the last transition temperature was shifted to higher temperature, when the buffer solution was manipulated. The mean values in strongly binding states were Tm1=52.9±0.7°C, Tm2=57.9±0.7°C, Tm3=63.7±1.0°C and Tm4=67.8±0.7°C, but the last transition increased to higher temperature depending on the Pi analogue.

Thermal and Molecular Characterization of Aspergillus awamori Glucoamylase Catalytic and Starch-Binding Domains

Aspergillus awamori glucoamylase catalytic domain, linker, and starch-binding domain, the first and third expressed from yeast, have molecular masses of 56.2, 12.6 and 12.9 kDa, respectively, as determined by MALDI-TOF mass spectroscopy, and have 10.2, 73.2 and 7.0 % (w/w) carbohydrate, respectively, showing overglycosylation by yeast. Unfolding of the starch-binding domain monitored by circular dichroism is reversible at pH 6.0- 8.0, with the unfolding T m and AH increasing from 49.7 to 58.5 °C and from 284 to 351 kJ/mol, respectively, as pH decreases from 8.0 to 6.0. The catalytic domain unfolds irreversibly at pH 7.5, producing a single asymmetric endotherm by differential scanning calorimetry, with T m and ΔH at a 1 °C/min heating rate being 60.9 °C and 1720 kJ/mol, but with both increasing as the heating rate increases. This suggests that unfolding is partially under kinetic control, while various tests show that it does not follow a simple two-state irreversible model. Values of AH from calculated solvent-accessible surface areas of unglycosylated catalytic and starch-binding domains are about 100 kJ/mol lower than experimentally determined AH values of the corresponding glycosylated domains, showing the effect of glycosylation on unfolding enthalpies.

Characterization of Mucor pusillus rennin expressed in Pichia pastoris: enzymic, spectroscopic and calorimetric studies.

The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to beta-sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two-state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimensional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993) J. Mol. Biol. 221, 1295-1309].

Two-state irreversible thermal denaturation of muscle creatine kinase

Thermal denaturation of creatine kinase from rabbit skeletal muscle has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were independent of protein concentration, but strongly temperature scanning rate-dependent. It has been shown that thermal denaturation of creatine kinase satisfies the previously proposed validity criteria for the two-state irreversible model [Kurganov et al., Biophys. Chem.70 (1997) 125]. The energy activation value has been calculated to be 461.0±0.7 kJ/mol.

Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

We report on a differential scanning calorimetry study of native purple membranes under the following solvent conditions: 50 mM carbonate-bicarbonate, 100 mM NaCl, pH 9.5 and 190 mM phosphate, pH 7.5. The calorimetric transitions for bacteriorhodopsin denaturation are highly scanning-rate dependent, which indicates that the thermal denaturation is under kinetic control. This result is confirmed by a spectrophotometric study on the kinetics of the thermal denaturation of this protein. The calorimetric data at pH 9.5 conform to the two-state irreversible model. Comments are made regarding the information obtainable from differential scanning calorimetry studies on bacteriorhodopsin denaturation and the effect of irreversibility on the stability of membrane proteins.

Differential scanning calorimetric study of carboxypeptidase B, procarboxypeptidase B and its globular activation domain

High-sensitivity differential scanning calorimetry has been applied to the study of porcine pancreatic carboxypeptidase B, the proenzyme and its 81-residue activation domain. The thermal study has been carried out over a range of scan rates, ionic strengths and pH values. The thermal unfolding of the isolated activation domain has been found to be reversible and corresponds to that of a typical compact globular structure, with melting temperatures higher than those of the enzyme and proenzyme. Both proteins, on the other hand, undergo an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. The denaturation of the enzyme at pH 7.5 and the proenzyme at pH 7.5 and 9.0 follows the two-state irreversible model [Sánchez-Ruiz, J.M., López-Lacomba, J.L., Cortijo, M. & Mateo, P.L. (1988) Biochemistry 27, 1648-1652]. Thus the kinetic constants and activation parameters of the denaturation process could be obtained and compared to those for other proteins, particularly those of the closely related carboxypeptidase A system.