Studies on the effect of temperature on the activity and stability of cyanobacterial ADP-glucose pyrophosphorylase

The effect of temperature on the activity and stability of ADPglucose pyrophosphorylase from Anabaena PCC 7120 was studied. Experimental optima temperatures were found around 37-40 degrees C or 42-45 degrees C, depending on the absence or the presence of allosteric effectors in the assay medium, respectively. In the range of temperature where the enzyme is stable, curved Arrhenius plots were obtained, indicating a transition temperature between 9 and 12 degrees C. Since these results were observed for both the forward and reverse reaction, with two different sets of substrates and two entirely different assay procedures, it seems unlikely that the effect can be on any component of the system other than the enzyme itself. Results suggest that cyanobacterial ADPglucose pyrophosphorylase undergoes conformational changes at different temperatures, rendering structures with different catalytic efficiencies. The different structures of the enzyme were visualized by emission fluorescence. ADPglucose pyrophosphorylase was irreversibly inactivated when exposed to temperatures above 40 degrees C. Inactivation was dependent on temperature and followed first order kinetics. The substrate, ATP, and the allosteric effectors, 3PGA and Pi, effectively protected the enzyme against thermal inactivation. Protection afforded by ATP was affected by MgCl2. These results suggest that the binding of the effectors to the enzyme resulted in conformational changes of the protein, rendering structures more stable to temperature treatments. Similar structures could be adopted by the enzyme in different environments, since the higher stability was observed in media containing either high ionic strength or high hydrophobicity.     

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.     

Conformational transitions of antibodies induced by hapten binding

The conformational changes of antibody structure induced by hapten molecule binding were investigated by means of thermal perturbation difference spectroscopy. The studies of the free rabit anti-dinitrophenyl antibodies show the conformational transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. Binding of the hapten molecules induces a similar transition to that which occurs between the two temperature dependent states of the free antibody. In contrast to our previous results with anti-dansyl rabbit antibodies the dinitrophenyl lysine stabilizes the "low temperature" native state of the protein. The investigation of the MOPC-315 mouse immunoglobulin A myeloma protein possessing anti-dinitrophenyl activity indicates no conformational transition at temperatures between 25 and 35 degrees C and only a small decrease of tyrosine exposure induced by the hapten binding. Our present and previous results indicate that most of the free immunoglobulins exist in two native conformational states which have a small difference in free energy. Hapten binding causes the transition in equilibrium between the two states towards the one of better binding. It is possible that this transition is necessary but not sufficient step for inducing the effector function of antibodies.     

Temperature studies of glyceraldehyde-3-phosphate dehydrogenase binding to liposomes using fluorescence technique.

Interaction of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase with negatively charged liposomes was investigated as a function of temperature. This interaction affects the temperature-dependent conformational transition in the enzyme and exerts stabilizing effect on the protein structure. It can be seen from the fluorescence quenching experiments that the accessibility of tryptophanyl residues and isoindol probe fluorophores (covalently bound with the protein amino groups) for a dynamic quencher, acrylamide, is altered upon binding. This accessibility represented by effective quenching constant (Keff) strongly depends on temperature for unmodified enzyme and for the enzyme adsorbed on liposomes, it is nearly constant over a wide range of temperatures.     

Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.     

Effects of temperature on diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart. Aspects of the kinetics, stability, and quarternary structure of the enzyme.

A temperature-dependent conformational change of the active DPN-linked isocitrate dehydrogenase was observed. When initial reaction kinetic data were examined between 35 and 5 degrees, the Hill number (n) varied from 2 at higher to n approaching unity at lower temperatures, with an inflection point at 17 degrees. The presence of manganous isocitrate in the incubation media shifted the transition temperature for enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoate) from 8-16 degrees. These temperature-dependent transitions were paralleled by progressive changes in sedimentation velocities from s20, w of 10.4 at 25 degrees to 7.3 at 10 degrees as measured by active band centrifugation. The linear Arrhenius plot for apparent V max and the constancy of S0.5 for the substrate manganous isocitrate between 35 and 5 degrees suggest that this temperature-dependent conformational change may not be solely related to manganous isocitrate. Further indications of equilibria between different species of enzyme in solution and effects of substrates and cofactors on conformation came from studies of specific activity of enzyme diluted into buffers at 3 and 25 degrees. Dilution to concentrations between 10 and 25 mum enzyme resulted in relatively rapid protein concentration-dependent inactivation which could be prevented and fully reversed by manganous isocitrate. No further substantial inactivation was found subsequent to this phase at 25 degrees. Lowering the temperature of the dilution buffer to 3 degrees favored formation of enzyme species exhibiting a further time and pH-dependent loss of activity which became independent of protein concentration below 7 mum enzyme. The rate of cold inactivation was reduced by raising the ionic strength of the buffer and its progress could be arrested by manganous isocitrate; however, the substrate did not restore the original activity.     

A comparative molecular dynamics study of thermophilic and mesophilic ribonuclease HI enzymes

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

Temperature dependence of dynamics of hydrated myoglobin. Comparison of force field calculations with neutron scattering data

Molecular dynamics is used to probe the atomic motions of the carboxy-myoglobin protein as a function of temperature. Simulations of 150 picoseconds in length are carried out on the protein at 20, 60, 100, 180, 220, 240, 260, 280, 300, 320 and 340 K. The simulations attempt to mimic neutron scattering experiments very closely by including a partial hydration shell around the protein. Theoretical elastic, quasielastic and inelastic neutron scattering data are derived from the trajectories and directly compared with experiment. Compared to experiment, the simulation-derived elastic scattering curves show a decrease in intensity as a function of the scattering wavevector, q2. The inelastic and quasielastic spectra show that the inelastic peak is shifted to lower frequency than the experimental value, while quasielastic behavior is in good agreement with experiment. This suggests that the theoretical model is too flexible in the harmonic limit (low temperature), but accurately reproduces high-temperature behavior. Time correlation functions of the intermediate scattering function are determined. At low temperature there is one fast decay process, and at high temperatures there is an additional slow relaxation process that is due to quasielastic scattering. The average atomic fluctuations show that the protein behaves harmonically at low temperatures. At approximately 210 K, a glass-like transition in atomic fluctuations is seen. Above the transition temperature, the atomic fluctuations exhibit both harmonic and anharmonic behavior. Comparison of protein mobility behavior with experiment indicate the fluctuations derived from simulations are larger in the harmonic region. However, the anharmonic region agrees very well with experiment. The anharmonicity is large at all temperatures, with a gradual monotonic increase from 0.5 at 20 K to greater than 0.7 at 340 K without a noticeable change at the glass transition temperature. Heavy-atom dihedral transitions are monitored as a function of temperature. Trends in the type of dihedral transitions that occur with temperature are clearly visible. Dihedral transitions involving backbone atoms occur only above the glass transition temperature. The overall protein behavior results suggest that at low temperatures there is purely vibrational motion with one fast decay process, and above the glass transition temperature there is more anharmonic motion with a fast and a slower relaxation process occurring simultaneously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unzipping mechanism of the double-stranded DNA unwinding by a hexameric helicase: quantitative analysis of the rate of the dsDNA unwinding, processivity and kinetic step-size of the Escherichia coli DnaB helicase using rapid quench-flow method

Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining approximately 11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(+/- 0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25 degrees C unwinds approximately 291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P = 0.89(+/- 0.03), little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.     

A Comparative Molecular Dynamics Study of Thermophilic and Mesophilic Ribonuclease HI Enzymes

Abstract

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

The molecular basis of the temperature- and pH-induced conformational transitions in elastin-based peptides

Elastin undergoes an inverse temperature transition and collapses at high temperatures in both simulation and experiment. We investigated a pH-dependent modification of this transition by simulating a glutamic acid (Glu)-substituted elastin at varying pHs and temperatures. The Glu-substituted peptide collapsed at higher temperature than the unsubstituted elastin when Glu was charged. The charge effects could be reversed by neutralization of the Glu carboxyl groups at low pH, and in that case the peptide collapsed at a lower temperature. The collapse was accompanied by the formation of beta-turns and short distorted beta-sheets. Formation of contacts between hydrophobic side chains drives the collapse at high temperature, but interactions between water and polar groups (Glu and main chain) can attenuate this effect at high pH. The overall competition and balance of the polar and nonpolar groups determined the conformational states of the peptide. Water hydration contributed to the conformational transition, and the peptide and its hydration shell must be considered. Structurally, waters near polar residues mainly formed hydrogen bonds with the protein atoms, while waters around the hydrophobic side chains tended to be parallel to the peptide groups to maximize water-water interactions.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

Neutron scattering reveals the dynamic basis of protein adaptation to extreme temperature

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 A) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.     

Adjustment of conformational flexibility of glyceraldehyde-3-phosphate dehydrogenase as a means of thermal adaptation and allosteric regulation

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Thermotoga maritima (TmGAPDH) is a thermostable enzyme (T (m) = 102 degrees C), which is fully active at temperatures near 80 degrees C but has very low activity at room temperature. In search for an explanation of this behavior, we measured the conformational flexibility of the protein by hydrogen-deuterium exchange and compared the results with those obtained with GAPDH from rabbit muscle (RmGAPDH). At room temperature, the conformational flexibility of TmGAPDH is much less than that of RmGAPDH, but increases with increasing temperature and becomes comparable to that of RmGAPDH near the physiological temperature of Thermotoga maritima. Using the available three-dimensional structures of the two enzymes, we compared the B factors that reflect the local mobility of protein atoms. The largest differences in B factors are seen in the coenzyme and NAD binding regions. The likely reason for the low activity of TmGAPDH at room temperature is that the motions required for enzyme functions are restricted. The findings support the idea of "corresponding states" which claims that over the time span of evolution, the overall conformational flexibility of proteins has been preserved at their corresponding physiological temperatures.     

Temperature-dependent vibrational and conformational dynamics of photosystem II membrane fragments from spinach investigated by elastic and inelastic neutron scattering

Vibrational and conformational protein dynamics of photosystem II (PS II) membrane fragments from spinach were investigated by elastic and inelastic incoherent neutron scattering (EINS and IINS). As to the EINS experiments, the average atomic mean square displacement values of PS II membrane fragments hydrated at a relative humidity of 57% exhibit a dynamical transition at ~230K. In contrast, the dynamical transition was absent at a relative humidity of 44%. These findings are in agreement with previous studies which reported a "freezing" of protein mobility due to dehydration (Pieper et al. (2008) Eur. Biophys. J. 37: 657-663) and its correlation with an inhibition of electron transfer from Q(A)(-) to Q(B) (Kaminskaya et al. (2003) Biochemistry 42, 8119-8132). IINS spectra of a sample hydrated at a relative humidity of 57% show a distinct Boson peak at ~7.5meV at 20K, which shifts towards lower energy values upon temperature increase to 250K. This unexpected effect is interpreted in terms of a "softening" of the protein matrix along with the onset of conformational protein dynamics as revealed by the EINS experiments. Information on the density of vibrational states of pigment-protein complexes is important for a realistic calculation of excitation energy transfer kinetics and spectral lineshapes and is often routinely obtained by optical line-narrowing spectroscopy at liquid helium temperature. The data presented here demonstrate that IINS is a valuable experimental tool in determining the density of vibrational states not only at cryogenic, but also at nearly physiological temperatures up to 250K. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Effect of molecular confinement on internal enzyme dynamics: frequency domain fluorometry and molecular dynamics simulation studies

The tryptophanyl emission decay of the mesophilic beta-galactosidase from Aspergillus oryzae free in buffer and entrapped in agarose gel is investigated as a function of temperature and compared to that of the hyperthermophilic enzyme from Sulfolobus solfataricus. Both enzymes are tetrameric proteins with a large number of tryptophanyl residues, so the fluorescence emission can provide information on the conformational dynamics of the overall protein structure rather than that of the local environment. The tryptophanyl emission decays are best fitted by bimodal Lorentzian distributions. The long-lived component is ascribed to close, deeply buried tryptophanyl residues with reduced mobility; the short-lived one arises from tryptophanyl residues located in more flexible external regions of each subunit, some of which are involved in forming the catalytic site. The center of both lifetime distribution components at each temperature increases when going from the free in solution mesophilic enzyme to the gel-entrapped and hyperthermophilic enzyme, thus indicating that confinement of the mesophilic enzyme in the agarose gel limits the freedom of the polypeptide chain. A more complex dependence is observed for the distribution widths. Computer modeling techniques are used to recognize that the catalytic sites are similar for the mesophilic and hyperthermophilic beta-galactosidases. The effect due to gel entrapment is considered in dynamic simulations by imposing harmonic restraints to solvent-exposed atoms of the protein with the exclusion of those around the active site. The temperature dependence of the tryptophanyl fluorescence emission decay and the dynamic simulation confirm that more rigid structures, as in the case of the immobilized and/or hyperthermophilic enzyme, require higher temperatures to achieve the requisite conformational dynamics for an effective catalytic action and strongly suggest a link between conformational rigidity and enhanced thermal stability.     

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

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

Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity

DeCuevas et al. [J. Cell Biol. 116 (1992) 957-965] demonstrated by circular dichroism spectroscopy for the kinesin stalk fragment that shifting temperature from 25 to 30 degrees C caused a conformational transition. To gain insight into functional consequences of such a transition, we studied the temperature dependence of a full-length kinesin by measuring both the velocity of microtubule gliding across kinesin-coated surfaces and microtubule-promoted kinesin ATPase activity in solution. The corresponding Arrhenius plots revealed distinct breaks at 27 degrees C, corroborating the temperature-dependent conformational transition for a motility-competent full-length kinesin. Microtubules were found to glide up to 45 degrees C; at higher temperatures, kinesin was irreversibly damaged.     

Evidence that the conformational stability of 'aged' organophosphate-inhibited cholinesterase is altered

In order to determine whether a structural modification at the active center of cholinesterase may alter the conformational stability of the enzyme we compared the urea-induced unfolding of the tetrameric form of non-inhibited and irreversibly inhibited human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). We studied enzyme inhibited by methanesulfonyl fluoride, diisopropylfluorophosphonate (DFP) and racemic soman. DFP- and soman-inhibited cholinesterases are converted spontaneously into non-reactivable forms called 'aged' enzymes through a process involving dealkylation of the bound organophosphate residue. The unfolding was followed by transverse urea-gradient polyacrylamide electrophoresis at various temperatures ranging from 0 to 60 degrees C. Unfolding of cholinesterase appears to be a complex process. The denaturation patterns showed that partially unfolded states are thermodynamically unstable, but that several intermediates are involved; the lifetime of these depends on the temperature at which electrophoreses are carried out. Cholinesterase inhibited by methanesulfonyl fluoride behaved like the non-inhibited enzyme. On the other hand, small but significant differences in stability between non-inhibited and aged enzymes were observed. Whatever the temperature, the urea concentration at the mid-point of transition was always greater for aged enzyme than for the non-inhibited enzyme. In addition, aged enzymes showed more complex denaturation patterns at the lower temperatures (under 20 degrees C). These findings suggest that the overall stability of aged-cholinesterases is slightly increased as compared with the stability of non-inhibited or methanesulfonyl fluoride-inhibited enzymes. The denaturation pattern obtained at 0 degree C for soman-inhibited cholinesterase under non-aging conditions (inhibition at 0 degree C, pH 10.7) was similar to that of non-inhibited enzyme at this temperature, although splitting in two of the denaturation curve over the transition zone reflects the heterogeneity of soman-inhibited enzyme. The slight difference in denaturation behavior between these species may be due to stereoisomerism in soman. The differences in electrophoretic behavior and apparent stability observed between non-inhibited and aged enzymes were interpreted as the result of a conformational change induced by the dealkylation reaction of enzyme-inhibitor conjugates.