Equimolar Mixture of 2,2,2-Trifluoroethanol and 4-Chloro-1-butanol is a Stronger Inducer of Molten Globule State: Isothermal Titration Calorimetric and Spectroscopic Studies

A mixture of 4-chloro-1-butanol and 2,2,2-Trifluoroethanol (TFE) has been used to generate Molten globule (MG) state of structurally homologous but functionally different proteins bovine α-lactalbumin and hen egg-white lysozyme. The thermal denaturation was done using UV–Visible spectroscopy. From UV–Visible profile, thermal transition was not observed beyond a particular concentration. There was an indication of molten globule state in case of α-lactalbumin from circular dichroism experiments. By intrinsic tryptophan fluorescence, acrylamide and potassium iodide quenching, 8-anilino-naphthalene sulfonic acid (ANS) binding and energy transfer studies the presence of molten globule state was confirmed. Quantitative characterization of MG state and determining the binding thermodynamics of ANS to the MG state was done using Isothermal Titration Calorimetry (ITC). Results show that α-lactalbumin exists in MG state at a particular concentration but lysozyme does not show features of MG state.     

Conformational stability of LYLA1, a synthetic chimera of human lysozyme and bovine α-lactalbumin

LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca²⁺-binding site and helix C) of bovine α-lactalbumin has been inserted. The equilibrium unfolding of this hybrid protein has been examined by circular dichroism and tryptophan fluorescence techniques. The reversible denaturation process induced by temperature or by addition of chemical denaturant is three-state in the case of apo-LYLA1 and two-state in the presence of Ca²⁺. The Ca²⁺-bound form of the chimera exhibits higher stability than both wild-type lysozyme and α-lactalbumin. The stability of the apo-form, however, is intermediate between that of the parent molecules. Unfolding of apo-LYLA1 involves an intermediate state that becomes populated to a different extent under various experimental conditions. Combination of circular dichroism with bis-ANS fluorescence experiments has permitted us to characterize the acid state of LYLA1 as a molten globule. Furthermore our results strongly suggest the presence of multiple denatured states depending on external conditions. Received: 24 April 1996 / Accepted: 4 September 1996     

Equilibrium and kinetic studies on folding of canine milk lysozyme

Thermal and chemical unfolding studies of the calcium-binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped-flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo-canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s(-1). Furthermore, the results show that, unlike its drastic effect on the stability, Ca(2+)-binding only marginally affects the refolding kinetics. During the refolding process of apo-CL non-native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non-native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

Unfolding and Refolding of Bovine α-Crystallin in Urea and Its Chaperone Activity

We undertook an unfolding and refolding study of α_L-crystallin in presence of urea to explore the breakdown and formation of various levels of structure and to find out whether the breakdown of various levels of structure occurs simultaneously or in a hierarchal manner. We used various techniques such as circular dichroism, fluorescence spectroscopy, light scattering, polarization to determine the changes in secondary, tertiary, and quaternary structure. Unfolding and refolding occurred through a number of intermediates. The results showed that all levels of structure in α_L-crystallin collapsed or reformed simultaneously. The intermediates that occurred in the 2–4 M urea concentration range during unfolding and refolding differed from each other in terms of the polarity of the tryptophan environment. The ANS binding experiments revealed that refolded α_L-crystallin had higher number of hydrophobic pockets compared to native one. On the other hand, polarity of these pockets remained same as that of the native protein. Both light scattering and polarization measurements showed smaller oligomeric size of refolded α_L-crystallin. Thus, although the secondary structural changes were almost reversible, the tertiary and quaternary structural changes were not. The refolded α_L-crystallin had more exposed hydrophobic sites with increased binding affinity. The refolded form also showed higher chaperone activity than native one. Since the refolded form was smaller in oligomeric size, some buried hydrophobic sites were available. The higher chaperone activity of lower sized oligomer of α_L-crystallin again revealed that chaperone activity was dependent on hydrophobicity and not on oligomeric size.     

Refolding intermediate of guanidine hydrochloride denatured aminoacylase

The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.     

The kinetics of G-CSF folding

The folding kinetics of G-CSF were determined by trp-fluorescence and far-UV circular dichroism. Folding and unfolding was achieved by rapid dilution and mixing of the denaturant, GdnHCl. G-CSF is a four-helical bundle protein with two long loops between the first and second helices and between the third and fourth helices. The entire conformational change expected by fluorescence was observed by stopped-flow technology, but due to rapid refolding kinetics only a portion was observed by circular dichroism. G-CSF contains two trp residues, and their contribution to the fluorescent-detected kinetics were deciphered through the use of single-site trp mutants. The trp moieties are probes of the local conformation surrounding their environment. One trp at residue 118 is located within the third helix while the other trp at residue 58 is part of the long loop between the first and second helices. The refolding results were most consistent with the following mechanism: U <--> I(1) <--> I(2) <--> N; where U represents the unfolded protein, I(1) represents intermediate state 1, I(2) represents intermediate state 2, and N represents the native state. I(1) is characterized as having approximately one-half of the native-like helical structure and none of the native-like fluorescence. I(2) has 100% of the native helical structure and most of the trp-118 and little of the trp-58 native-like fluorescence. Thus refolding occurs in distinct stages with half of the helix forming first followed by the remaining half of the helix including the third helix and finally the loop between the first and second helices folds.     

Kinetics of folding and unfolding of goat alpha-lactalbumin

The equilibrium unfolding and the kinetic folding and unfolding of goat alpha-lactalbumin (GLA) were studied by near- and far-ultraviolet circular dichroism (CD) and by stopped-flow fluorescence spectroscopy. Specifically, the influence of environmental conditions such as pH and Ca2+ binding was examined. Compared to the apo-form, the Ca2+-bound form was found to be strongly stabilized in equilibrium conditions at pH 7.5 and 25 degrees C. The kinetics of the refolding of apo-GLA show a major change of fluorescence intensity during the experimental dead-time, but this unresolved effect is strongly diminished in holo-GLA. In both cases, however, the chevron plots can adequately be fitted to a three-state model. Moreover, double-mix stopped-flow experiments showed that the native state (N) is reached through one major pathway without the occurrence of alternative tracks. In contrast to the homologous bovine alpha-lactalbumin (BLA), the compactness of GLA is strongly influenced by the presence of Ca2+ ions. Unlike the two-state transition observed in guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation experiments at higher pH, an equilibrium intermediate state (I) is involved in denaturation at pH 4.5. In the latter case, analysis of the kinetic data makes clear that the intermediate and the unfolded states (U) show practically no Gibbs free energy difference and that they are in rapid equilibrium with each other. A possible explanation for these variations in stability and in folding characteristics with pH could be the degree of protonation of His107 that directly influences non-native interactions. Variation of environmental conditions and even small differences in sequence, therefore, can result in important effects on thermodynamic and folding parameters.     

Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate

The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared.     

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.     

Structurally homologous all beta-barrel proteins adopt different mechanisms of folding

Acidic fibroblast growth factors from human (hFGF-1) and newt (nFGF-1) (Notopthalamus viridescens) are 16-kDa, all beta-sheet proteins with nearly identical three-dimensional structures. Guanidine hydrochloride (GdnHCl)-induced unfolding of hFGF-1 and nFGF-1 monitored by fluorescence and far-UV circular dichroism (CD) shows that the FGF-1 isoforms differ significantly in their thermodynamic stabilities. GdnHCl-induced unfolding of nFGF-1 follows a two-state (Native state to Denatured state(s)) mechanism without detectable intermediate(s). By contrast, unfolding of hFGF-1 monitored by fluorescence, far-UV circular dichroism, size-exclusion chromatography, and NMR spectroscopy shows that the unfolding process is noncooperative and proceeds with the accumulation of stable intermediate(s) at 0.96 M GdnHCl. The intermediate (in hFGF-1) populated maximally at 0.96 M GdnHCl has molten globule-like properties and shows strong binding affinity to the hydrophobic dye, 1-Anilino-8-naphthalene sulfonate (ANS). Refolding kinetics of hFGF-1 and nFGF-1 monitored by stopped-flow fluorescence reveal that hFGF-1 and nFGF-1 adopts different folding mechanisms. The observed differences in the folding/unfolding mechanisms of nFGF-1 and hFGF-1 are proposed to be either due to differential stabilizing effects of the charged denaturant (Gdn(+) Cl(-)) on the intermediate state(s) and/or due to differences in the structural interactions stabilizing the native conformation(s) of the FGF-1 isoforms.     

Folding-unfolding equilibrium and kinetics of equine beta-lactoglobulin: equivalence between the equilibrium molten globule state and a burst-phase folding intermediate

The denaturant-induced equilibrium unfolding transition of equine beta-lactoglobulin was investigated by ultraviolet absorption, fluorescence, and circular dichroism (CD) spectra. An equilibrium intermediate populates at moderate denaturant concentrations, and its CD spectrum is similar to that of the molten globule state previously observed for this protein at acid pH [Ikeguchi, M., Kato, S., Shimizu, A., and Sugai, S. (1997) Proteins: Struct., Funct., Genet. 27, 567-575]. The unfolding and refolding kinetics were also investigated by the stopped-flow CD and fluorescence. A significant change in the CD intensity was observed within the dead time of measurements (25 ms) when the refolding reaction was initiated by diluting the urea-unfolded protein solution, indicating the transient accumulation of the folding intermediate. The CD spectrum of this burst-phase intermediate agrees well with that of the molten globule state at acid pH. The stability of the burst-phase intermediate was also estimated from the urea-concentration dependence of the burst-phase amplitude, and it shows a fair agreement with that of the equilibrium intermediate. These results indicate that the molten globule state of equine beta-lactoglobulin populates at moderate urea concentration as well as at acid pH and it is equivalent with the kinetic folding intermediate.     

Ca(II)- and Tb(III)-induced stabilization and refolding of anticoagulation factor I from the venom of Agkistrodon acutus

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF I, holo-ACF I, and Tb(3+)-reconstituted ACF I in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism. Metal ions were found to increase the structural stability of ACF I against GdnHCl and thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF I and Tb(3+)-ACF I is a two-state process with no detectable intermediate state(s), whereas the GdnHCl-induced unfolding/refolding of holo-ACF I in the presence of 1 mM Ca(2+) follows a three-step transition, with intermediate state a (Ia) and intermediate state b (Ib). Ca(2+) ions play an important role in the stabilization of the Ia and Ib states. The decalcification of holo-ACF I shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, whereas the reconstitution of apo-ACF I with Tb(3+) ions shifts the initial zone of denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.0 M GdnHCl) at which refolding from the fully denatured state of apo-ACF I to the Ib state of holo-ACF I or to the native state of Tb(3+)-ACF I can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF I, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ions-induced refolding provide evidence that the compact Tb(3+)-binding region forms first, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Unfolding and refolding of leucoagglutinin (PHA-L), an oligomeric lectin from kidney beans (Phaseolus vulgaris)

The unfolding and refolding of Phaseolus vulgaris Leucoagglutinin, a homotetrameric legume lectin, was studied at pH 2.5 and 7.2 using fluorescence, far- and near-UV circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalene sulfonate (ANS) binding and FPLC techniques. This protein was found to refold even at pH 2.5 and also exhibited high refolding yield around 60% at pH 2.5 and 85% at pH 7.2. The refolding at pH 2.5 takes place with the formation of a dimeric intermediate. Although the hydrodynamic radius of the completely renatured protein and the dimer at pH 2.5 was found to be same, the ANS binding as well as far-UV CD spectra of the two were different. The denaturation kinetics at pH 2.5 followed single exponential pattern with the rate of denaturation being independent of protein concentration. The renaturation kinetics on the other hand was dependent on the protein concentration providing further evidence of an intermediate state during refolding. From these experiments the folding pathway of the protein at pH 2.5 was proposed.     

Equilibrium and kinetics of the folding and unfolding of canine milk lysozyme

The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Chemical Unfolding of Enolase from Saccharomyces cerevisiae Exhibits a Three-State Model

Enolase is a multifunctional protein that participates in glycolysis and gluconeogenesis and can act as a plasminogen receptor on the cell surface of several organisms, among other functions. Despite its participation in a variety of biological and pathophysiological processes, its stability and folding/unfolding reaction have not been fully explored. In this paper we present, the urea and GdnHCl-induced denaturation of enolase studied by means of fluorescence and circular dichroism spectroscopies. We found that enolase unfolds through a highly reversible pathway, populating a stable intermediate species in a range of experimental conditions. The refolding reaction also exhibits an intermediate state that might have a slightly more compact conformation compared to the unfolding intermediate. The thermodynamic parameters associated with the unfolding reaction are presented and discussed.     

Kinetic studies of the unfolding-refolding of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of the native protein, but devoid of enzyme activity. The fluorescence emission spectrum of this intermediate was determined. Only the slow phase was detected for the unfolding process; it was not attributable to proline isomerization. Several models were assumed, and simulated kinetics derived from these models were compared with the experimental results. A plausible one accounting for most of the data is proposed.     

The folding of staphylococcal nuclease in the presence of methanol or guanidine thiocyanate

The effect of methanol on the folding of staphylococcal nuclease has been investigated. Equilibrium thermal unfolding transitions were monitored by fluorescence emission. The transition was very sensitive to the presence of methanol (at pH 7.0), the Tm decreased from above 50 degrees C for aqueous solution to below 0 degree C for 70% methanol. The transitions were fully reversible and conformed to two-state behavior. A linear relationship was observed between the hydrophobicity of the solvent and both the Tm and the change in delta G for unfolding. The effect of pH on the transition in 50% methanol at 0 degree C was essentially the same as for aqueous solution, with a cooperative transition in the vicinity of apparent pH (pH*) 4. The unfolding transition was determined as a function of guanidine thiocyanate in aqueous and 50% methanol solvents. The midpoints of the transitions were 0.30 and 0.20 M, respectively, at 2.1 degrees C. The kinetics of folding at 0 degree C were compared in aqueous, 50% methanol and 0.30 M guanidine thiocyanate solvents, by monitoring changes in the tryptophan fluorescence intensity. Triphasic kinetics for refolding in both aqueous and 50% methanol solutions were observed in stopped-flow experiments. In both solvent systems the slowest phase is ascribed to proline isomerization. The kinetics of refolding were monitored at subzero temperatures in 50% methanol at pH* 7.0 in manual mixing experiments. Biphasic kinetics were observed at temperatures between 0 and -35 degrees C. A third, faster phase, was inferred from the missing amplitude. The energies of activation were 20.0 and 17.2 kcal mol-1, respectively, for the two slower phases. At -33.8 degrees C, the observed pseudo first-order rate constants were 1.2 x 10(-3) and 2.1 x 10(-5) s-1. At temperatures above -35 degrees C, the sum of the observed amplitudes was essentially constant at 70-75% of the expected total amplitude. At lower temperatures the amplitude of the refolding reaction decreased, and the native state was not formed (unless the temperature was increased), due to the formation of a trapped intermediate state. This intermediate has circular dichroism and fluorescence properties consistent with a compact state with some molten globule characteristics.     

Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein.