Exposure of thiol groups in the heat-induced denaturation of β-lactoglobulin

Protein aggregates can be stabilised by disulphide bridges. The whey protein β-lactoglobulin (β-lac) contains a disulphide bridge and a free cysteine that are shielded from the solvent by an α-helix. These groups are important in the thiol–disulphide exchange that occurs during aggregation and gelation of β-lac. Replica exchange molecular dynamics simulations show that the exposure mechanism is very different for the two buried groups. While melting of the α-helix enhances exposure of the free cysteine, it does not for the buried bridge. These findings shed light on the molecular mechanism of the first step of β-lac denaturation and aggregation.     

Thermal and acid denaturation of bovine lens α-crystallin

The chaperone-like protein α-crystallin is a ～35 subunit hetero-oligomer consisting of αA and αB subunits in a 3:1 molar ratio and has the function of maintaining eye lens transparency. We studied the thermal denaturation of α-crystallin by differential scanning calorimetry (DSC), circular dichroism (CD), and dynamic light scattering (DLS) as a function of pH. Our results show that between pH 7 and 10 the protein undergoes a reversible thermal transition. However, the thermodynamic parameters obtained by DSC are inconsistent with the complete denaturation of an oligomeric protein of the size of α-crystallin. Accordingly, the CD data suggest the presence of extensive residual secondary structure above the transition temperature. Within the pH range from 4 to 7 the increased aggregation propensity around the isoelectric point (pI ～ 6) precludes observation of a thermal transition. As pH decreases below 4 the protein undergoes a substantial unfolding. The secondary structure content of the acid-denatured state shows little sensitivity to heating. We propose that the thermal transition above pH 7 and the acid-induced transition at ambient temperature result in predominant denaturation of the αB subunit. Although the extent of denaturation of the αA subunit cannot be estimated from the current data, the existence of a native-like conformation is suggested by the preserved association of the subunits and the chaperone-like activity. A key difference between the thermal and the acid denaturation is that the latter is accompanied by dissociation of αB subunits from the remaining αA-oligomer, as supported by DLS studies.     

On the course of thermal denaturation of deoxyribonucleic acids after γ-irradiation

Summary The changes which are caused by action of-irradiation onDNAs of various origin were followed by spectrophotometric method at differing thermal denaturation curves. It was found that all measured bacterialDNAs as well asDNA isolated from calf thymus, irradiated by exposures higher that 5×10⁴ R, produced significantly decreasedT _m values with concomittantly decreased hyperchromic effect and changed transition intervals obtained in 10^–2 M sodium phosphate, 10^–3 M EDTA medium at pH 7. It was also observed that higher-irradiation exposures caused the loss of renaturation ability ofEscherichia coli DNA.Abbreviations used DNA deoxyribonucleic acid - G guanine - C cytosine - E ₂₆₀ extinction (absorbancy) measured at 260m - T _m the temperature corresponding to the midpoint of the absorbance rise - 2/3 the transition interval of the denaturation curve corresponding to the temperature interval between 17–83% of the total absorbancy increment of the denaturation curve - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7) - PE 10^–2 M sodium phosphate buffer [molarity related to (PO₄)] - PE 10^–3 M EDTA, pH 7. 1.000 ml prepared as follows: 0.608 g NaH₂PO₄.2 H₂O, 0.218 g Na₂HPO₄.12 H₂O, 0.372 g disodium salt of EDTA, 1.0 ml 1 M NaOH. Concentration of Na ions — 0.02 M.     

Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).     

Thermal denaturation and structural changes of α-helical proteins in keratins

To gain insight into the thermal stability of intermediate filaments and matrix in the biological composite structure of α-keratins, the thermal denaturation performance of human hair fibers was investigated by Modulated Differential Scanning Calorimetry (MDSC) in the dry and the wet state. Denaturation enthalpy ΔH(D) in water was found to be independent of heating rate (11.5J/g) and to be approximately double as high as in the dry state (5.2J/g). The lower enthalpy (dry) and its dependency on heating rate are attributed to effects of pyrolysis. The stepwise change of reversing heat capacity ΔC(p) marks the denaturation process as a classic two-stage transition. The increase of ΔC(p) with heating rate reflects a continuous shift of the nature of the denaturation of the α-helical material, first, into random coil and then towards random β-materials for lower heating rates. Denaturation temperatures follow Arrhenius relationships with heating rate, yielding activation energies of 416kJ/mol (dry) and 263kJ/mol (wet), respectively. A decrease of activation energy (wet) for high heating rates supports the hypothesis of systematic changes of the pathway of denaturation.     

Environmental-induced acquisition of nuptial plumage expression: a role of denaturation of feather carotenoproteins?

Several avian species show a bright carotenoid-based coloration during spring and following a period of duller coloration during the previous winter, despite carotenoids presumably being fully deposited in feathers during the autumn moult. Carotenoid-based breast feathers of male linnets (Carduelis cannabina) increased in hue (redness), saturation and brightness after exposing them to outdoor conditions from winter to spring. This represents the first experimental evidence showing that carotenoid-based plumage coloration may increase towards a colourful expression due to biotic or abiotic environmental factors acting directly on full-grown feathers when carotenoids may be fully functional. Sunlight ultraviolet (UV) irradiation was hypothesized to denature keratin and other proteins that might protect pigments from degradation by this and other environmental factors, suggesting that sunlight UV irradiation is a major factor in the colour increase from winter to spring. Feather proteins and other binding molecules, if existing in the follicles, may be linked to carotenoids since their deposition into feathers to protect colourful features of associated carotenoids during the non-breeding season when its main signalling function may be relaxed. Progress towards uncovering the significance of concealment and subsequent display of colour expression should consider the potential binding and protecting nature of feather proteins associated with carotenoids.     

Why is the denaturation process of super-helical DNA so uncooperative?

A theoretical model is proposed for the helix-coil transition in circular covalently closed DNA. In the melting interval the topological constrains for such a molecule are considered to affect both the inner state of the melted regions and the spatial configuration of the whole molecule ("writhing"). A good agreement is achieved between the theoretically calculated and experimentally obtained parameters of the denaturation process. A conclusion is drawn, that at the early stages of thermodenaturation (approximately till the transition midpoint) the effect of topological constrains is realised mainly through the writhing of the molecule. The denatured regions exist in the form of relaxed loops.     

Effect of the lyotropic series of anions on denaturation and renaturation of 20β-hydroxysteroid dehydrogenase

The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20β-hydroxysteroid dehydrogenase (17,20β,21-trihydroxysteroid:NAD⁺ oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denaturated enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the inactivation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1–2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.     

A folding model of α-lactalbumin deduced from the three-state denaturation mechanism

It has been shown that α-lactalbumin undergoes a three-state denaturation, involving a helical intermediate state, on treatment with guanidine hydrochloride. The unfolding of the protein and the characteristics of the intermediate state are examined by means of circular dichroism, difference spectra and pH-jump measurements to investigate the temperature dependence and kinetic properties of the unfolding and refolding, the pH dependence of the transition between the intermediate and the fully unfolded states, and the effect of disulphide bond reduction on the stabilization of the intermediate.The results show that the long-range specific interactions such as specific electrostatic interactions and disulphide linkages are not important for stabilizing the intermediate, and that the transition between the intermediate and the fully unfolded states is extremely rapid (a relaxation time of less than one millisecond) and may correspond to the helix-coil transition of a polypeptide backbone. On the other hand, the activation parameters of the transition between the native and the intermediate states have suggested that the final stabilization by charge-pair interactions is preceded by hydrophobic interactions in the process of going from the intermediate to the native state.The mechanism of folding of the protein is discussed, and the folding process from the fully unfolded to the native state is apparently divided into at least three main steps: (1) the formation of incipient helical structures dictated by local interactions; (2) the packing of the helical segments accompanied with hydrophobic interactions; (3) the final stabilization by the electrostatic interactions. The relevance to the current theoretical results on protein folding is also discussed.     

Kinetic study of the thermal denaturation of a hyperthermostable extracellular α-amylase from Pyrococcus furiosus

Hyperthermophilic enzymes are of industrial importance and interest, especially due to their denaturation kinetics at commercial sterilisation temperatures inside safety indicating time–temperature integrators (TTIs). The thermal stability and irreversible thermal inactivation of native extracellular Pyrococcus furiosus α-amylase were investigated using differential scanning calorimetry, circular dichroism and Fourier transform infrared spectroscopy. Denaturation of the amylase was irreversible above a T_m of approximately 106 °C and could be described by a one-step irreversible model. The activation energy at 121 °C was found to be 316 kJ/mol. Using CD and FT-IR spectroscopy it was shown that folding and stability greatly increase with temperature. Under an isothermal holding temperature of 121 °C, the structure of the PFA changes during denaturation from an α-helical structure, through a β-sheet structure to an aggregated protein. Such data reinforces the use of P. furiosus α-amylase as a labile species in TTIs.     

The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background

The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B.

Methodology/Principal Findings

As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212–216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers.

Conclusions/Significance

Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B'' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation.     

Role of charged residues in stabilization of Pyrococcus horikoshii CutA1, which has a denaturation temperature of nearly 150 °C

The CutA1 protein from Pyrococcus horikoshii (PhCutA1), a hyperthermophile, has an unusually high content of charged residues and an unusually high denaturation temperature. To elucidate the role of ion-ion interactions in protein stability, mutant proteins of PhCutA1 in which charged residues were substituted by noncharged residues were comprehensively examined. The denaturation temperatures (T(d)) for 13 of 53 examined mutant proteins were higher than that of the wild-type (148.5 °C at pH 7.0), among which E99Q had the highest T(d) at 154.9 °C. R25A had the largest decrease in T(d) among single mutants at ΔT(d) = -12.4 °C. The average decrease in T(d) of Lys or Arg mutants was greater than that of Glu or Asp mutants, and the average change in T(d) (ΔT(d)) of 21 Glu mutants was negligible, at 0.03 ± 2.05 °C. However, the electrostatic energy (-159.3 kJ·mol(-1)) of PhCutA1 was quite high, compared with that of CutA1 from Escherichia coli (-9.7 kJ·mol(-1)), a mesophile. These results indicate that: (a) many Glu and Asp residues of PhCutA1 should be essential for highly efficient interactions with positively charged residues and for generating high electrostatic energy, although they were forced to be partially repulsive to each other; (b) the changes in stability of mutant proteins with a T(d) value of ~140-150 °C were able to be explained by considering factors important for protein stability and the structural features of mutant sites; and (c) these findings are useful for the design of proteins that are stable at temperatures > 100 °C.     

Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45 degrees C, of single molecules entrapped within the reverse micelles (0.031 min(-1)), was higher than in aqueous solution (0.007 min(-1)) or in the presence of normal micelles (0.020 min(-1)). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53 degrees C, were 0.317 and 0.342 min(-1), respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.     

Thermal denaturation: is solid-state fermentation really a good technology for the production of enzymes?

The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system. The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 x 10(59) h(-1) and 364,070 Jmol(-1), respectively. These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter. In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time. The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 degrees C, which occurred at the lower two airflow rates. A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale. It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation. This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products.     

Monitoring denaturation behaviour and comparative stability of DNA triple helices using oligonucleotide–gold nanoparticle conjugates

Gold nanoparticle labels, combined with UV-visible optical absorption spectroscopic methods, are employed to probe the temperature-dependent solution properties of DNA triple helices. By using oligonucleotide–nanoparticle conjugates to characterize triplex denaturation, for the first time triplex to duplex melting transitions may be sensitively monitored, with minimal signal interference from duplex to single strand melting, for both parallel and antiparallel triple helices. Further, the comparative sequence-dependent stability of DNA triple helices may also be examined using this approach. Specifically, triplex to duplex melting transitions for triplexes formed using oligonucleotides that incorporate 8-aminoguanine derivatives were successfully monitored and stabilization of both parallel and antiparallel triplexes following 8-aminoguanine substitutions is demonstrated.     

Thermal and chemical denaturation of Colocasia esculenta tuber agglutinin from α2β2 to unfolded state

The major tuber storage protein of Colocasia esculenta, is a monocot mannose-binding, widely used dietary lectin, containing two polypeptides of 12.0 and 12.4 kDa. By both gel filtration and dynamic light scattering at pH 7.2, the lectin has a α₂β₂ form of apparent molecular mass of 48.2 kDa and a hydrodynamic radius of 6.1 ± .2 nm; however, at pH 3, it migrates as αβ and has a reduced hydrodynamic radius of 4.6 ± .3 nm. Our circular dichroism spectroscopy studies show that the lectin retains approximately 100% of its secondary structure between pH 2–8, going down to ~90% for extreme acidic/alkaline conditions. The fluorescence emission maxima of 346 to 350 nm for pH 4 to 10 show that the tryptophan residues are relatively exposed. The unfolding is a simple two-state process, N₄ ? 4U, as seen in our denaturation scan profiles. These denaturation profiles, monitored separately by fluorescence, far-UV CD, and near-UV CD, are completely super imposable. Analyses of these profiles provide an estimate of several thermodynamic parameters at each guanidinium chloride concentration, including the melting temperature T_g, which is 346.9 K in 0 M, but lowers to 321.8 K in 3.6 M. Dimeric and tetrameric interfaces observed in the crystal structure for the same protein are used to rationalize solution data in some detail.     

Does beta-lactoglobulin denaturation occur via an intermediate state?

The denaturation of beta-lactoglobulin (BLG) in the presence of urea and GuHCl has been investigated at different pH values with various spectroscopic techniques. The equilibrium denaturation free energy values, obtained by linearly extrapolating the data to vanishing denaturant (DeltaG(D)(H2O)), are compared and discussed. The fit of the spectroscopic data monitoring the denaturation of BLG has been approached, at first, with a two-state model that describes the protein transition from the folded state (at each pH and in the absence of denaturant) to the denatured state, but in particular, along the GuHCl denaturation pathway some evidence is found of the presence of an intermediate state. Time-resolved fluorescence experiments performed on the BLG-ANS (1-anilino-8-naphthalenesulfonate) complex help to understand the results. Fluorescence polarization anisotropy (FPA) measurements accompanying the denaturation process show the presence of a fast rotational diffusion of the ANS probe, and the data are interpreted in terms of local fluctuations of a still structured tract of the denatured protein where the probe is bound.     

Comparison of denaturation of tryptophan synthase α-subunits from Escherichia coli,Salmonella typhimurium, and an interspecies hybrid

Guanidine hydrochloride-induced denaturation and thermal denaturation of three kinds of tryptophan synthase α subunit have been compared by circular dichroism measurements. The three α subunits are from Escherichia coli, Salmonella typhimurium, and an interspecies hybrid in which the C-terminal domain comes from E. coli (α-2 domain) and the N-terminal domain comes from S. typhimurium (α-1 domain). Analysis of denaturation by guanidine hydrochloride at 25 °C showed that the α-2 domain of S. typhimurium was more stable than the α-2 domain of E. coli, but the α-1 domain of S. typhimurium was less stable than the α-1 domain of the E. coli protein; overall, the hybrid protein was slightly less stable than the two original proteins. It is concluded that the stability to guanidine hydrochloride denaturation of each of the domains of the interspecies hybrid is similar to the stability of the domain of the species from which it originated. The E. coli protein was more stable to thermal denaturation than the other proteins near the denaturation temperature, but the order of their thermal stability was reversed at 25 °C and coincided with that obtained from guanidine hydrochloride-induced denaturation.     

β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation

Conversion of the rod-like tobacco mosaic virus (TMV) virions into “ball-like particles” by thermal denaturation at 90–98?°C had been described by R.G. Hart in 1956. We have reported recently that spherical particles (SPs) generated by thermal denaturation of TMV at 94–98?°C were highly stable, RNA-free, and water-insoluble. The SPs were uniform in shape but varied widely in size (53–800?nm), which depended on the virus concentration. Here, we describe some structural characteristics of SPs using circular dichroism, fluorescence spectroscopy, and Raman spectroscopy. It was found that the structure of SPs protein differs strongly from that of the native TMV and is characterized by coat protein subunits transition from mainly (about 50%) α-helical structure to a structure with low content of α-helices and a significant fraction of β-sheets. The SPs demonstrate strong reaction with thioflavin T suggesting the formation of amyloid-like structures.