Pressure-temperature-induced denaturation of yeast phosphoglycerate kinase, a double-domain protein.

Pressure-induced denaturation of yeast phosphoglycerate kinase was studied at various temperatures, as a model double-domain protein, using intrinsic fluorescence, 4th derivative absorbance, CD, and DSC. A thermodynamic transition intermediate was observed in the pressure-denaturation, as was reported for the cold denaturation. From the different response of Trp and Tyr residues, as monitored by fluorescence and 4th derivative absorbance changes, the C-terminal domain carrying all the Trp residues seemed to exert structural changes at relatively lower pressure. A further structural change involving both domains was observed at higher pressures. The two-step changes occurred almost simultaneously during heat denaturation.     

Stabilization of Mikamycin B Lactonase

Myoglobin (Mb) purified from fast skeletal muscle of bluefin tuna Thunnus thynnus orientalis was subjected to thermal treatment, and the denaturation profiles were examined by thermodynamic analysis. Based on the ellipticity or helical content obtained by circular dichroism (CD) spectrometry, it was found that denaturation of tuna Mb consisted of three steps, and that slight structural changes of Mb started below 20 °C. However, major structural changes were observed at around 58 and 72 °C. Differential scanning calorimetry (DSC) analysis revealed a similar but somewhat different thermal denaturation profile of Mb. In comparison with the denaturing profiles of whale Mb under the same conditions, the thermal stability of tuna Mb was found to be much lower. In the modeled tertiary structures of these Mbs, they were roughly similar to each other, though minor conformational differences were recognized and the total energy was found to be lower for tuna Mb.     

Insights into protein–polysorbate interactions analysed by means of isothermal titration and differential scanning calorimetry

Therapeutic proteins formulated as liquid solutions at high protein concentration are very sensitive to chemical and physical degradation. Especially avoiding the formation of protein aggregates is very crucial for product quality. In order to stabilize the colloidal properties of protein therapeutics various excipient are used. Especially the detergents polysorbate 20 and 80 are common. However, the mechanism upon which the detergents protect the protein from aggregation is not really known. The present study investigates the interaction of polysorbate 20 and 80 with different proteins: lysozyme, bovine serum albumin (BSA) and an immunoglobulin. The interaction and binding of the detergents to the proteins is investigated by isothermal titration calorimetry (ITC). From ITC the thermodynamic parameters (ΔH: change in enthalpy, ΔS: entropy and ΔG: free energy) upon binding are derived as well as the binding constant K _a. The thermal stability of the proteins in the presence of the detergent is assessed by differential scanning calorimetry (DSC). The results show that both detergents bind to BSA with K _a between 8 and 12 × 10³ M⁻¹ with ΔH −50 to −60 kJ/mol (25°C). One to two detergent molecules bind to BSA. The presence of both detergents induces a weak stabilisation of the thermal denaturation properties of BSA. However, the interaction of polysorbate 20 and 80 with lysozyme and the immunoglobulin is quite negligible. The presence of the detergents up to a concentration of 2 mM has no impact on the heat capacity curve neither a destabilisation nor a stabilisation of the native conformation is observed.     

Predicting changes in protein thermostability brought about by single- or multi-site mutations

Background  

An important aspect of protein design is the ability to predict changes in protein thermostability arising from single- or multi-site mutations. Protein thermostability is reflected in the change in free energy (ΔΔG) of thermal denaturation.     

The effect of some osmolytes on the activity and stability of mushroom tyrosinase

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) fromAgaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40°C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obeye the first order law. Inactivation rate constant (k_inact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of k_inact(0.7×10⁻⁴s⁻¹) in comparison with their absence (2.5×10⁻⁴s⁻¹). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodyanamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.     

Differential Scanning Calorimetrie Study of the Thermal Denaturation of Almond β-Glucosidase

The thermal denaturation of almond β-glucosidase [EC 3.2.1.21] was studied by differential scanning calorimetry. The shape of the DSC trace was highly dependent on pH; two peaks were observed between pH 6–8, but only one peak between pH 4–5. All of the DSC curves were resolved into three components according to the model of independent two-state processes, and the thermodynamic parameters for the denaturation were evaluated. The dependence of the shape of DSC curves was accounted for mainly by the rapid changes of denaturation enthalpy and denaturation temperature of the third component in the acidic pH region.     

Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by differential scanning calorimetry

The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. In this study, we have used differential scanning calorimetry (DSC) and circular dichroism (CD) to measure the enthalpy change and melting temperature of the apo-CRP and CRP complexes with cAMP or DNA sequences lac, gal, and palindromic ICAP. DSC and CD measurements showed irreversible thermal denaturation process of CRP. Enthalpy of dissociation of the protein–DNA complex, as measured by DSC, depends on the DNA sequence. The thermal transition of the protein in CRP-DNA complexes, measured by CD, indicates that the protein stability in the complex is also DNA sequence-dependent.     

Thermal stability of wild type and disulfide bridge containing mutant of poplar plastocyanin

A comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning calorimetry and optical spectroscopic techniques. For wild type plastocyanin the transition temperature, determined from the calorimetric profiles, is 62.7 degrees C at the scan rate of 60 degrees C/h, whereas for the mutant it is reduced to 58.0 degrees C. In both cases, the endothermic peak is followed by an exothermic one at higher temperatures. The unfolding process monitored by optical absorption at 596 nm also reveals a reduced thermal stability of the mutated plastocyanin compared to the wild type protein, with transition temperatures of 54.8 and 58.0 degrees C, respectively. For both proteins, the denaturation process was found to be irreversible and dependent on the scan rate preventing the thermodynamic analysis of the unfolding process. In parallel, small conformational changes between wild type and mutant plastocyanin emerge from fluorescence spectroscopy measurements. Here, a difference in the interaction of the two proteins between the microenvironment surrounding the fluorophores and the solvent was proposed. The destabilization observed in the disulfide containing mutant of plastocyanin suggests that the double mutation, Ile21Cys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink.     

Thermodynamic description of oligonucleotide self-association in DNA concatamer structures

A scheme of equilibrium formation of concatamers by two different oligonucleotides has been considered. It is shown that in the general case, the dependence of the concentration of oligonucleotide components on temperature cannot be found in analytical form. Therefore, it is impossible to find the thermodynamic parameters of concatamer formation (ΔH ⁰, ΔS ⁰) and melting temperatures by analyzing the profiles of thermal denaturation of oligonucleotide complexes. An algorithm for numerical solution of implicit dependences has been developed. A number of approaches are considered that simplify the analysis of heat denaturation curves for concatamer complexes. It is shown that the dependence of the efficiency of concatamerization on temperature can be described analytically when duplex fragments have close stability and there is no cooperativity at the oligonucleotide junction. In this case, the dependence of melting temperature on thermodynamic parameters and oligonucleotide concentration has the same form as in the case of the duplex structure formed by a pair of non-self-complementary oligonucleotides. The ability of various model approaches to describe the experimental curves of concatamer heat denaturation is evaluated. For concatamer structures used as signal amplifiers in DNA hybridization analysis, a function is introduced that shows the relative contribution of a concatamer of given length to the magnitude of signal amplification. The dependence of the maximum of this function on the concentration of oligonucleotides, the thermodynamic characteristics of their complexes, and temperature has been determined. It is shown by the gel retardation assay that the function of the length distribution of concatamers qualitatively correlates with the experimental dependences.     

Three easy pieces

BackgroundDifferential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of temperature. There are, however, circumstances that preclude a complete analysis of DSC data. The most common ones are irreversible denaturation transitions or transitions that take place at temperatures that are beyond the temperature limit of the instrument. Even for a protein that undergoes reversible thermal denaturation, the extrapolation of the thermodynamic data to lower temperatures, usually 25 °C, may become unreliable due to difficulties in the determination of ΔC_p.MethodsThe combination of differential scanning calorimetry and isothermal chemical denaturation allows reliable thermodynamic analysis of protein stability under less than ideal conditions.Results and conclusionsThis paper demonstrates how DSC can be used in combination with chemical denaturation to address three different scenarios: 1) estimation of an accurate ΔC_p value for a reversible denaturation using as a test system the envelope HIV-1 glycoprotein gp120; 2) determination of the Gibbs energy of stability in the region in which thermal denaturation is irreversible using HEW lysozyme at different pH values; and, 3) determination of Gibbs energy of stability for a thermostable protein, thermolysin. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.     

Effect of Ficoll 70 on thermal stability and structure of creatine kinase

The effect of Ficoll 70 on the thermal stability and structure of creatine kinase (CK) was studied using far-UV CD spectra and intrinsic fluorescence spectra. The thermal transition curves monitored by CD spectra were fitted to a two-state model using a modified form of the van’t Hoff equation to obtain the transition temperature (T _m) and enthalpy change (ΔH _u) of thermally induced denaturation of CK in the absence and presence of Ficoll 70. An increase in T _m with constant ΔH _u was observed with increasing Ficoll 70 concentration, suggesting that Ficoll 70 enhances the thermal stability of CK. Fluorescence spectral measurements confirmed this protective effect of Ficoll 70 on CK structure. In addition, we observed a crowding-induced compaction effect on the structure of both native state and thermally denatured state of CK in the presence of Ficoll 70, which is more obvious on the structure of the denatured ensemble compared to that of the native ensemble. Our observations qualitatively accord with the predictions of previously proposed crowding theory for the effect of intermolecular excluded volume on protein stability and structure. These findings imply that the effects of macromolecular crowding are essential to our understanding of protein folding and unfolding occurring in vivo.     

ESR Spectroscopy Investigation of the Denaturation Process of Soybean Peroxidase Induced by Guanidine Hydrochloride, DMSO or Heat

The involvement of protein denaturation and/or misfolding processes in the insurgence of several diseases raises the interest in structural dynamic studies of proteins. The use of nitroxide spin labels with electron paramagnetic resonance is a powerful tool for detecting structural changes in proteins. In the present study, we apply this strategy to soybean peroxidase (SBP), a protein characterised by high thermal and structural stability, and we propose a simple method to analyse the anisotropy changes of the protein system and to relate them with the structural changes induced by protein unfolding. We examined the effect of temperature, guanidine hydrochloride and dimethylsulfoxide on the stability of SBP and looked for correlations between the ESR results and the experimental findings obtained by other techniques, reported in the literature. The agreement between data obtained through different strategies supports the validity and reliability of the ESR approach to protein unfolding.     

Effect of Glucose on the Lactoferrin’s Conformation and its Effect on MC 3T3-E1 Cell Proliferation

The objective of this research was to investigate the influence of glucose on bovine lactoferrin’s (LF) conformation, thermodynamic stability and osteoblastic cell proliferation. The conformation and thermodynamic stability of LF was detected by spectroscopic and differential scanning calorimetry (DSC). The osteoblastic cell proliferation of LF at physiological concentrations (100 μg/ml) was measured by BrdU incorporation. The binding constant between glucose and LF is KSV = 5 × 10⁻³, and Tyr residues of LF were located in a more hydrophobic environment, while Trp residues were located in a more hydrophilic environment. LF with glucose had increased α-helix and β-sheet contents by 6 and 14 %, respectively. It showed a two-step denaturation of LF. There was a gradual changs in the denaturation temperature and the calorimetric enthalpies (ΔHcal) with a growing concentration of glucose. It has also revealed that glucose dose dependently reduced the ability of LF to increase MC 3T3-E1 cell proliferation. Increasing the binding with glucose, LF might cause to change its native state, which reduced the stimulation activity of osteoblasts cell proliferation.     

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

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

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c ₆ reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c ₆ does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I. This revised version was published online in June 2006 with corrections to the Cover Date.     

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (～100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day^?1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.     

Effects of subclass change on the structural stability of chimeric,humanized, and human antibodies under thermal stress

To address how changes in the subclass of antibody molecules affect their thermodynamic stability, we prepared three types of four monoclonal antibody molecules (chimeric, humanized, and human) and analyzed their structural stability under thermal stress by using size‐exclusion chromatography, differential scanning calorimetry (DSC), circular dichroism (CD), and differential scanning fluoroscopy (DSF) with SYPRO Orange as a dye probe. All four molecules showed the same trend in change of structural stability; the order of the total amount of aggregates was IgG1 < IgG2 < IgG4. We thus successfully cross‐validated the effects of subclass change on the structural stability of antibodies under thermal stress by using four methods. The T_h values obtained with DSF were well correlated with the onset temperatures obtained with DSC and CD, suggesting that structural perturbation of the CH2 region could be monitored by using DSF. Our results suggested that variable domains dominated changes in structural stability and that the physicochemical properties of the constant regions of IgG were not altered, regardless of the variable regions fused.     

The Use of the Free Metal – Temperature ‘Phase Diagrams’ for Studies of Single Site Metal Binding Proteins

Typical physico-chemical studies of metal binding proteins are usually aimed at determination of the metal binding constant K for a native protein (K _n), while the significance of the K value for the thermally denatured protein (K _u) is usually underestimated. Meanwhile, metal binding induced shift of thermal denaturation transition of a single site metal binding protein is defined by K _n to K _u ratio, implying that knowledge of both K values is required for full characterization of the system. In the present work, the most universal approach to the studies of single site metal binding proteins, namely construction of a protein “phase diagram” in coordinates of free metal ion concentration – temperature, is considered in detail. The detailed algorithm of construction of the phase diagrams along with underlying mathematic procedures developed here may be of use for studies of other simple protein-target type systems, where target represents low molecular weight ligand. Analysis of the simplest protein-ligand system reveals that thermodynamic properties of apo-protein dictate the maximal possible increase of its affinity to any simple ligand upon thermal denaturation of the protein. Experimental and general problems coupled with the use of the phase diagrams are discussed.