Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin

The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.     

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA.

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Synergistic inhibition of acid-induced protein denaturation by trehalose and NaCl: Thermodynamic and kinetic studies

It is known that trehalose and sodium chloride (NaCl) can both effectively inhibit acid-induced protein denaturation, but the thermodynamic and kinetic behaviors of acid-induced protein unfolding synergistically inhibited by trehalose and NaCl are unclear. In this study, the synergistic inhibition effects of trehalose and NaCl on the acid-induced unfolding of ferricytochrome c were studied at pH 2.0. Thermodynamic parameters were firstly derived based on fluorescence spectroscopic data. Then, kinetic behaviors were studied using stopped-flow fluorescence spectroscopy. It was found that the kinetics of the acid-induced protein unfolding transformed from a triphasic process (i.e., fast, intermediate and slow phases) into a biphasic one (i.e., intermediate and slow phases) and then a single slow phase process with increasing either trehalose or NaCl concentration in the mixture. The rate constants for all the unfolding phases change slightly, while the amplitudes for the fast and intermediate phases diminish greatly with increasing the concentration of trehalose or NaCl. This clearly indicates that the mixture of trehalose and NaCl could synergistically inhibit acid-induced protein unfolding by reducing the extent of protein conformational changes, thus inducing a stable molten-globule state at higher concentrations of the agents.     

Mechanisms of the multiphasic kinetics in the folding and unfolding of globular proteins

The multiphasic kinetics of the protein folding and unfolding processes are examined for a “cluster model” with only two thermodynamically stable macroscopic states, native (N) and denatured (D), which are essentially distributions of microscopic states. The simplest kinetic schemes consistent with the model are: N-(fast) → I-(slow) → D for unfolding and N ← (fast)-D₂ ← (slow)-D₁ for refolding. The fast phase during the unfolding process can be visualized as the redistribution of the native population N to I within its free energy valley. Then, this population crosses over the free energy barrier to the denatured state D in the slow phase. Therefore, the macrostate I is a kinetic intermediate which is not stable at equilibrium. For the refolding process, the initial equilibrium distribution of the denatured state D appears to be separated into D₁ and D₂ in the final condition because of the change in position of the free energy barrier. The fast refolding species D₂ is due to the “leak” from the broadly distributed D state, while the rest is the slow refolding species D₁, which must overpass the free energy barrier to reach N. At an early stage of the folding process the amino acid chain is considered to be composed of several locally ordered regions, which we call clusters, connected by random coil chain parts. Thus, the denatured state contains different sizes and distributions of clusters depending on the external condition. A later stage of the folding process is the association of smaller clusters. The native state is expressed by a maximum-size cluster with possible fluctuation sites reflecting this association. A general discussion is given of the correlation between the kinetics and thermodynamics of proteins from the overall shape of the free energy function. The cluster model provides a conceptual link between the folding kinetics and the structural patterns of globular proteins derived from the X-ray crystallographic data.     

Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins

Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins (∼ 400-800 s^− 1), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10⁴ with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.     

Effects of salt on the thermal stability of human plasma high-density lipoprotein

High-density lipoproteins (HDL) mediate cholesterol removal and thereby protect against atherosclerosis. Mature spherical HDL contain the apolar lipid core and polar surface of proteins and phospholipids. Earlier, we showed that the structural integrity of HDL is modulated by kinetic barriers that prevent spontaneous protein dissociation and lipoprotein fusion and rupture. To determine the role of electrostatic interactions in the kinetic stability of mature HDL, here we analyze the effects of salt and pH on their thermal denaturation. In low-salt buffer at pH 5.7-7.7, HDL are highly thermostable. Increasing the salt concentration from 0 to 0.3 M NaCl causes low-temperature shifts in the calorimetric HDL transitions of up to -14 degrees C. This salt-induced destabilization leads to protein unfolding below 100 degrees C, facilitating the first Arrhenius analysis of HDL denaturation by circular dichroism spectroscopy. In 150 mM NaCl, two kinetic phases in HDL protein unfolding are observed: a faster phase with an activation energy E(a,fast) < or =15 kcal/mol and a slower phase with an E(a,slow) = 50 +/- 7 kcal/mol. Gel electrophoresis and electron microscopic data suggest that the faster phase involves partial protein unfolding but no significant protein dissociation or changes in HDL size, while the slower phase involves complete protein unfolding, partial protein dissociation, and HDL fusion. Hence, the slower phase may resemble HDL remodeling and fusion by plasma enzymes during metabolism. Analysis of the effects of various salts, sucrose, and pH suggests that HDL destabilization by salt results from ionic screening of favorable short-range electrostatic interactions such as salt bridges. Consequently, electrostatic interactions significantly contribute to the high thermostability of HDL in low-salt solutions.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

Differential scanning calorimetric study of the thermal unfolding of beta-lactamase I from Bacillus cereus.

The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques. DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled. The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase. The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters. This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition. Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1,..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states. Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on.     

Counteraction of trehalose on urea-induced protein unfolding: Thermodynamic and kinetic studies

Urea-induced protein denaturation can be effectively inhibited by trehalose, but the thermodynamic and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on urea-induced unfolding of ferricytochrome c was studied. Thermodynamic parameters for the counteraction of trehalose were derived based on fluorescence spectroscopic data. Then the kinetics was emphatically investigated by stopped-flow fluorescence spectroscopy. Urea-induced unfolding of ferricytochrome c in 8.00 mol/L urea solution reveals two observable phases, including fast and slow phases following a burst phase. Trehalose has little influence on the burst phase amplitude. Nevertheless, the observable unfolding pathway is significantly affected by trehalose. At lower trehalose concentrations (<0.20 mol/L) in 8.00 mol/L urea, the unfolding pathways still keep to show two phases. However, the rate constant and amplitude for the fast phase diminish with increasing trehalose concentration. In contrast, the rate constant for the slow phase shows only a slight change with a significant increase of the amplitude. At higher trehalose concentrations (>0.30 mol/L), the unfolding pathway is transformed into a single slow phase. The rate constant and amplitude for the single phase also decrease with increasing trehalose concentration. The studies are expected to help our understanding of trehalose effects on protein stability.     

Phi-analysis at the experimental limits: mechanism of beta-hairpin formation

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 degrees Celsius, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first beta-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the beta-strands in the denatured state and that these interactions provide the driving force for folding of the first beta-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between beta1 and beta2 does a hydrogen bond form to stabilize the intervening turn, or the first beta-turn.     

Coarse-grained strategy for modeling protein stability in concentrated solutions. II: phase behavior

We use highly efficient transition-matrix Monte Carlo simulations to determine equilibrium unfolding curves and fluid phase boundaries for solutions of coarse-grained globular proteins. The model we analyze derives the intrinsic stability of the native state and protein-protein interactions from basic information about protein sequence using heteropolymer collapse theory. It predicts that solutions of low hydrophobicity proteins generally exhibit a single liquid phase near their midpoint temperatures for unfolding, while solutions of proteins with high sequence hydrophobicity display the type of temperature-inverted, liquid-liquid transition associated with aggregation processes of proteins and other amphiphilic molecules. The phase transition occurring in solutions of the most hydrophobic protein we study extends below the unfolding curve, creating an immiscibility gap between a dilute, mostly native phase and a concentrated, mostly denatured phase. The results are qualitatively consistent with the solution behavior of hemoglobin (HbA) and its sickle variant (HbS), and they suggest that a liquid-liquid transition resulting in significant protein denaturation should generally be expected on the phase diagram of high-hydrophobicity protein solutions. The concentration fluctuations associated with this transition could be a driving force for the nonnative aggregation that can occur below the midpoint temperature.     

Kinetically controlled refolding of a heat-denatured hyperthermostable protein

The thermal denaturation of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144 degrees C, corresponding to protein denaturation and complete unfolding, respectively, as shown by circular dichroism and fluorescence spectroscopy data. Calorimetric studies also showed that the denatured state did not refold to the native state unless the cooling temperature rate was very slow. Furthermore, previously denatured protein samples gave well-resolved denaturation transition peaks and showed enzymatic activity after 3 and 9 months of storage, indicating slow refolding to the native conformation over time.     

pH dependence of bacteriorhodopsin thermal unfolding

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.     

Folding under inequilibrium conditions as a possible reason for partial irreversibility of heat-denatured proteins: computer simulation study

Using computer simulations we have studied possible effects of heating and cooling at different scan rates on unfolding and refolding of macromolecules. We have shown that even the simplest two-state reversible transition can behave irreversibly when an unfavorable combination of cooling rate, relaxation time and activation energy of refolding occurs. On the basis of this finding we suppose that apparent irreversibility of some proteins denatured by heat may result from slow relaxation on cooling rather than thermodynamic instability and/or irreversible alterations of the polypeptide chain. Using this kinetic reversible two-state model, we estimated the effects of the scan rate and kinetic parameters of the macromolecule on its unfolding-refolding process. A few recommendations are suggested on how to reach maximal possible recovery after denaturation if refolding appears to be under kinetic control.     

Analysis of protein folding by fast protein liquid chromatography. Modular domain folding of gamma-II-crystallin from calf eye-lens

Fast protein liquid chromatography was effectively applied to analyse the folding mechanism of gamma-II-crystallin from calf eye-lens. The protein undergoes a bimodal folding/unfolding transition, according to a three-state model: N in equilibrium I in equilibrium D where N, I, and D stand for the native, intermediate and denatured states (R. Rudolph, R. Siebendritt, G. Nesslauer, A.K. Sharma & R. Jaenicke (1990) Proc. Natl. Acad. Sci. USA 87, 4625-4629). Using Superose 12 HR 10/30, the intermediate with the N-terminal domain intact, and the C-terminal domain unfolded, could be separated from the native protein. The N----I transition is sufficiently slow to allow kinetic measurements, following the variation of the respective peak-heights during denaturation/renaturation. The corresponding relaxation times are in agreement with kinetic data based on the change in fluorescence emission accompanying the N in equilibrium I transition.     

Folding thermodynamics and kinetics of the leucine-rich repeat domain of the virulence factor Internalin B

Although the folding of alpha-helical repeat proteins has been well characterized, much less is known about the folding of repeat proteins containing beta-sheets. Here we investigate the folding thermodynamics and kinetics of the leucine-rich repeat (LRR) domain of Internalin B (InlB), an extracellular virulence factor from the bacterium Lysteria monocytogenes. This domain contains seven tandem leucine-rich repeats, of which each contribute a single beta-strand that forms a continuous beta-sheet with neighboring repeats, and an N-terminal alpha-helical capping motif. Despite its modular structure, InlB folds in an equilibrium two-state manner, as reflected by the identical thermodynamic parameters obtained by monitoring its sigmoidal urea-induced unfolding transition by different spectroscopic probes. Although equilibrium two-state folding is common in alpha-helical repeat proteins, to date, InlB is the only beta-sheet-containing repeat protein for which this behavior is observed. Surprisingly, unlike other repeat proteins exhibiting equilibrium two-state folding, InlB also folds by a simple two-state kinetic mechanism lacking intermediates, aside from the effects of prolyl isomerization on the denatured state. However, like other repeat proteins, InlB also folds significantly more slowly than expected from contact order. When plotted against urea, the rate constants for the fast refolding and single unfolding phases constitute a linear chevron that, when fitted with a kinetic two-state model, yields thermodynamic parameters matching those observed for equilibrium folding. Based on these kinetic parameters, the transition state is estimated to comprise 40% of the total surface area buried upon folding, indicating that a large fraction of the native contacts are formed in the rate-limiting step to folding.     

Cooperative equilibrium transitions coupled with a slow annealing step explain the sharpness and hysteresis of collagen folding.

Heat and guanidinium-induced denaturation curves of collagen III and its fragments were fitted by theoretical models to explain the extreme sharpness and the hysteresis between unfolding and refolding. It was shown that a recently proposed kinetic model for collagen denaturation does not account for the observed steepness, with physically reasonable values of activation energy and frequency factors in the Arrhenius equation. The extreme slope, which amounts to 0.38 per centigrade for collagen III at the midpoint of its transition, can only be explained by a highly cooperative equilibrium model. The refolding curve is shifted to lower temperatures by 6 degrees C for collagen III and reversible unfolding matching the initial profile of the native protein is observed only after long-time annealing. A simple formalism is proposed by which experimental denaturation and refolding curves are quantitatively described. The transition proceeds via many cooperative steps with slightly different equilibrium constants for unfolding and refolding. Hysteresis and annealing are caused by very slow steps, which are probably connected with a rearrangement of misfolded regions. These slow steps disappear with decreasing size of collagen fragments and hysteresis is not found for collagen model peptides.     

Replacement of a conserved proline eliminates the absorbance-detected slow folding phase of iso-2-cytochrome c

As a test of the proline isomerization model, we have used oligonucleotide site-directed mutagenesis to construct a mutant form of iso-2-cytochrome c in which proline-76 is replaced by glycine [Wood, L. C., Muthukrishnan, K., White, T. B., Ramdas, L., & Nall, B. T. (1988) Biochemistry (preceding paper in this issue)]. For the oxidized form of Gly-76 iso-2, an estimate of stability by guanidine hydrochloride induced unfolding indicates that the mutation destabilizes the protein by 1.2 kcal/mol under standard conditions of neutral pH and 20 degrees C (delta G degrees u = 3.8 kcal/mol for normal Pro-76 iso-2 versus 2.6 kcal/mol for Gly-76 iso-2). The kinetics of folding/unfolding have been monitored by fluorescence changes throughout the transition region using stopped-flow mixing. The rates for fast and slow fluorescence-detected refolding are unchanged, while fast unfolding is increased in rate 3-fold in the mutant protein compared to normal iso-2. A new kinetic phase in the 1-s time range is observed in fluorescence-detected unfolding of the mutant protein. The presence of the new phase is correlated with the presence of species with an altered folded conformation in the initial conditions, suggesting assignment of the phase to unfolding of this species. The fluorescence-detected and absorbance-detected slow folding phases have been monitored as a function of final pH by manual mixing between pH 5.5 and 8 (0.3 M guanidine hydrochloride, 20 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Unfolding kinetics of glutathione reductase from cyanobacterium Spirulina maxima

The kinetics of the irreversible unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase, EC 1.6.4.2.) from cyanobacterium Spirulina maxima was studied at pH 7.0 and room temperature. Denaturation was induced by guanidinium chloride and the changes in enzyme activity, aggregation state, and tertiary structure were monitored. No full reactivation of enzyme was obtained, even after very short incubation times in the presence of denaturant. Reactivation plots were complex, showing biphasic kinetics. A very fast early event in the denaturation pathway was the dissociation of tetrameric protein into reactivatable native-like dimers, followed by its conversion into a nonreactivatable intermediary, also dimeric. In the final step of the unfolding pathway the latter was dissociated into denatured monomers. Fluorescence measurements revealed that denaturation of S. maxima glutathione reductase is a slow process. Release of the prostethic group FAD was previous to the unfolding of the enzyme. No aggregated species were detected in the unfolding pathway, dismissing the aggregation of denatured polypeptide chains as the origin of irreversibility. Instead, the transition between the two dimeric intermediates is proposed as the cause of irreversibility in the denaturation of S. maxima glutathione reductase. A value of 106.6 +/- 3 kJ mol(-1) was obtained for the activation free energy of unfolding in the absence of denaturant. No evidence for the native monomer in the unfolding pathway was obtained which suggests that the dimeric nature of glutathione reductase is essential for the maintenance of the native subunit conformation.