Dissociation and in vitro reconstitution of bovine liver uridine diphosphoglucose dehydrogenase. The paired subunit nature of the enzyme

Uridine diphosphoglucose dehydrogenase (EC 1.1.1.22: UDPglucose dehydrogenase) at pH 5.5-7.8 is a stable homohexamer of 305 +/- 7 kDa that does not undergo concentration-dependent dissociation at enzyme concentrations greater than 5 micrograms/mL. Chemical cross-linking of the native enzyme at varying glutaraldehyde concentrations yields dimers, tetramers, and hexamers; at greater than 2% (w/v) glutaraldehyde, plateau values of 21% monomers, 16% dimers, 5% tetramers, and 58% hexamers are obtained. Dissociation at acid pH (pH 2.3) or in 4-6 M guanidine hydrochloride leads to inactive monomers (Mr 52,000). Denaturation at increasing guanidine hydrochloride concentration reveals separable unfolding steps suggesting the typical domain structure of dehydrogenases holds for the present enzyme. At greater than 4 M guanidine hydrochloride complete randomization of the polypeptide chains is observed after 10-min denaturation. Reconstitution of the native hexamer after dissociation/denaturation has been monitored by reactivation and glutaraldehyde fixation. The kinetics may be described in terms of a sequential uni-bimolecular model, governed by rate-determining folding and association steps at the monomer level. Trimeric intermediates do not appear in significant amounts. Reactivation is found to parallel hexamer formation. Structural changes during reconstitution (monitored by circular dichroism) are characterized by complex kinetics, indicating the rapid formation of "structured monomers" (with most of the native secondary structure) followed by slow "reshuffling" prior to subunit association. The final product of reconstitution is indistinguishable from the initial native enzyme.     

Effects of low concentrations of guanidine . HCl on the reconstitution of lactic dehydrogenase from pig muscle in vitro. Evidence for guanidine binding to the native enzyme

The presence of low concentrations of guanidine . HCl has a pronounced effect on the overall rate of reactivation of lactic dehydrogenase from pig muscles after preceding dissociation and deactivation in various denaturants. The obseverd attenuation is a function of the amount of guanidine . HCl present during reconstitution. At a given guanidine concentration in the reactivation buffer the yield, but not the rate of reactivation, is influenced by the extent of denaturation caused initially in the process of deactivation and dissociation. As a possible explanation for the influence of guanidine . HCl on the kinetics of reconstitution, binding of the ligand to intermediates of folding and association is considered. This hypothesis is corroborated by the observation that guanidine . HCl in the relevant concentration range does bind to native lactic dehydrogenase without inactivating the enzyme or disrupting its quaternary structure. A kinetic model comprising guanidine binding to both the native enzyme and structured intermediates is proposed to describe the observed effects of guanidine . HCl on the rate of reactivation. In addition, the dissociation constants for guanidine binding to intermediates of reconstitution and to native lactic dehydrogenase are estimated.     

Mechanism and specificity of reconstitution of dimeric lactate dehydrogenase from Limulus polyphemus

D-Lactate dehydrogenase (EC 1.1.1.28) from Limulus polyphemus is a homodimer which is composed of identical subunits of Mr = 35 000. The enzyme may be reversibly denatured and dissociated at acid pH or in 6M guanidine X HCl. The sigmoidal time course of reactivation obeys a consecutive uni-bimolecular mechanism with k1 = 6 X 10(-4) S-1 and k2 = 1.3 X 10(-4) M-1 S-1 (20 degrees C) as first- and second-order rate constants. Cross-linking experiments with glutaraldehyde prove that reactivation and dimer formation run parallel. Joint "synchronous" reconstitution of the enzyme with dimeric porcine mitochondrial malate dehydrogenase (after denaturation in 6M guanidine X HCl) does not yield active hybrids. The unchanged kinetics of reactivation in the absence and presence of the prospective partner of hybridization prove that inactive hybrid intermediates may also be excluded. The absence of hybrids upon synchronous reconstitution of the two closely related dimeric NAD-dependent dehydrogenases clearly suggests that the assembly of nascent oligomeric proteins must be highly specific.     

Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 2. Reactivation of irreversibly denatured aggregates

Noncovalent aggregation is a side reaction in the process of reconstitution of oligomeric enzymes (e.g., lactic dehydrogenase) after preceding dissociation, denaturation, and deactivation. The aggregation product is of high molecular weight and composed of monomers which are trapped in a minium of conformational energy different from the one characterizing the native enzyme. This energy minimum is protected by a high activation energy of dissociation such that the aggregates are perfectly stable under nondenaturing conditions, and their degradation is provided only by applying strong denaturants, e.g., 6 M guanidine hydrochloride at neutral or acidic pH. The product of the slow redissolution process is the monomeric enzyme in its random configuration, which may be reactivated by diluting the denaturant under optimum conditions of reconstitution. The yield and the kinetics of reactivation of lactic dehydrogenase from pig skeletal muscle are not affected by the preceding aggregation-degradation cycle and are independent of different modes of aggregate formation (e.g., by renaturation at high enzyme concentration or heat aggregation). The kinetics of reactivation may be described by one single rate-determining bimolecular step with k2 = 3.9 x 10(4) M-1 s-1 at zero guanidine concentration. The reactivated enzyme consists of the native tetramer, characterized by enzymatic and physical properties identical with those observed for the enzyme in its initial native state.     

Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 +/- 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine X HCl concentrations is characterized by a two-step profile: at 0.4-0.8 M, partial unfolding is parallelled by inactivation; at 2.0-2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4-1.8 M guanidine X HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine X HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine X HCl, as monitored by the decrease of protein fluorescence, is fast (less than 8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (less than 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by tau = 29 +/- 3 min (20 degrees C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate-limiting for reactivation after long-term denaturation, are characterized by a high energy of activation (112 +/- 8 kJ/mol). The slow reactivation step is compatible in rate with the first-order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62-90].     

In vitro folding pathway of phage P22 tailspike protein.

The intracellular chain folding and association pathway of the thermostable, trimeric phage P22 tailspike endorhamnosidase has been the subject of a previous detailed study employing temperature-sensitive folding mutants. Recently, reconstitution of native tailspikes from completely unfolded polypeptides has been accomplished, providing a model system to compare protein folding pathways in vivo and in vitro. The in vitro reconstitution pathway of the protein after dilution from guanidine hydrochloride or acid-urea solutions at 10 degrees C was characterized by spectroscopic and hydrodynamic techniques, and may be summarized as an ordered sequence of folding, association, and folding reactions. Multiphasic folding of monomers was indicated by changes in circular dichroism and fluorescence, with a rate constant of k = 1.6 X 10(-3) s-1 for the slowest phase observed spectroscopically. Trimerization of structured monomers was followed by size-exclusion HPLC and was completed within 1.5 h at a protein concentration of 20 micrograms/mL. Although at this time trimers did not exchange subunits, they were readily dissociable by dodecyl sulfate in the cold. Formation of native, detergent-resistant trimers was only completed after 3 days of reconstitution at 10 degrees C. The reconstitution pathway of the tailspike protein closely resembles its intracellular maturation path. Thus, the in vitro reconstitution system, as a valid model of chain folding and association in vivo, should provide the tools to localize the steps or intermediates on the pathway that are the targets of temperature-sensitive folding mutations.     

Observation of multistate kinetics during the slow folding and unfolding of barstar.

The kinetics of the slow folding and unfolding reactions of barstar, a bacterial ribonuclease inhibitor protein, have been studied at 23(+/-1) degrees C, pH 8, by the use of tryptophan fluorescence, far-UV circular dichroism (CD), near-UV CD, and transient mixing (1)H nuclear magnetic resonance (NMR) spectroscopic measurements in the 0-4 M range of guanidine hydrochloride (GdnHCl) concentration. The denaturant dependences of the rates of folding and unfolding processes, and of the initial and final values of optical signals associated with these kinetic processes, have been determined for each of the four probes of measurement. Values determined for rates as well as amplitudes are shown to be very much probe dependent. Significant differences in the intensities and rates of appearance and disappearance of several resolved resonances in the real-time one-dimensional NMR spectra have been noted. The NMR spectra also show increasing dispersion of chemical shifts during the slow phase of refolding. The denaturant dependences of rates display characteristic folding chevrons with distinct rollovers under strongly native as well as strongly unfolding conditions. Analyses of the data and comparison of the results obtained with different probes of measurement appear to indicate the accumulation of a myriad of intermediates on parallel folding and unfolding pathways, and suggest the existence of an ensemble of transition states. The energetic stabilities of the intermediates estimated from kinetic data suggest that they are approximately half as stable as the fully folded protein. The slowness of the folding and unfolding processes (tau = 10-333 s) and values of 20.5 (+/-1.4) and 18 (+/-0.5) kcal mol(-)(1) for the activation energies of the slow refolding and unfolding reactions suggest that proline isomerization is involved in these reactions, and that the intermediates accumulate and are therefore detectable because the slow proline isomerization reaction serves as a kinetic trap during folding.     

Native-like intermediate on the folding pathway of Escherichia coli succinyl-CoA synthetase

The transition between the native and denatured states of the tetrameric succinyl-CoA synthetase from Escherichia coli has been investigated by circular dichroism, fluorescence spectroscopy, cross-linking by glutaraldehyde and activity measurements. At pH 7.4 and 25 degrees C, both denaturation of succinyl-CoA synthetase by guanidine hydrochloride and refolding of the denatured enzyme have been characterized as reversible reactions. In the presence of its substrate ATP, the denatured enzyme could be successfully reconstituted into the active enzyme with a yield of 71-100%. Kinetically, reacquisition of secondary structure by the denatured enzyme was rapid and occurred within 1 min after refolding was initiated. On the other hand, its reactivation was a slow process which continued up to 25 min before 90% of the native activity could be restored. Both secondary and quaternary structures of the enzyme, reconstituted in the absence of ATP, were indistinguishable from those of the native enzyme but the renatured protein was catalytically inactive. This observation indicates the presence of catalytically inactive tetramer as an intermediate in the reconstitution process. The reconstituted protein could be reactivated by ATP even 10 min after the reacquisition of the native secondary structure by the refolding protein. However, reactivation of the protein by ATP 60 min after the regain of secondary structure was significantly less, suggesting that rapid refolding and reassociation of the monomers into a native-like tetramer and reactivation of the tetramer are sequential events; the latter involving slow and small conformational rearrangements in the refolded enzyme that are likely to be associated with phosphorylation.     

Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations.     

Perturbation of folding and reassociation of lactate dehydrogenase by proline and trimethylamine oxide.

Investigations of protein-solute interactions typically show that osmolytes favor native conformations. In this study, the effects of representative compatible and counteracting osmolytes on the reactivation of lactate dehydrogenase from two different conformational states were explored. Contrary to expectations, proline and trimethylamine oxide inhibited both the initial time course and the extent of reactivation of lactate dehydrogenase from bovine heart following denaturation in guanidine hydrochloride, as well as following inactivation at pH 2.3. Reactivation of acid-dissociated porcine heart lactate dehydrogenase was inhibited by both proline and trimethylamine oxide (2 M). In all instances, trimethylamine oxide was the more effective inhibitor of reactivation. Analysis of the catalytic properties of the reactivating enzyme provided evidence that the molecular species that was enzymatically active during the initial stages of reactivation of acid-inactivated porcine heart lactate dehydrogenase reflects a non-native conformation. Proline and trimethylamine oxide stabilize polypeptides through exclusion from the polypeptide backbone; the inhibition of renaturation/reassociation described here is probably due to attenuation of this stabilizing influence through favorable interactions of the osmolytes with sidechains of residues that lie at the interfaces of the monomers and dimers that associate to form the active tetramer. In addition, these osmolytes may stabilize non-native intermediates in the folding pathway. The high viscosity of solutions containing more than 3 m proline was a major factor in the inhibition of reassociation of acid-dissociated porcine heart lactate dehydrogenase as well as other viscosity-dependent transformations that may occur during reactivation following unfolding in guanidine hydrochloride.     

Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

Reactivation of tetrameric porcine skeletal muscle lactic dehydrogenase after dissociation and extensive unfolding of the monomers by 6 M guanidine hydrochloride (Gdn . HCl) is characterized by sigmoidal kinetics, indicating a complex mechanism involving rate-limiting folding and association steps. For analysis of the association reactions, chemical cross-linking with glutaraldehyde may be used [Hermann, R., Jaenicke, R., & Rudolph, R. (1981) Biochemistry 20, 2195-2201]. The data clearly show that the formation of a dimeric intermediate is determined by a first-order folding reaction of the monomers with k1 = (8.0 +/- 0.1) x 10(-4) s-1. The rate constant of the association of dimers to tetramers which represents the second rate-limiting step on the pathway of reconstitution after guanidine denaturation, was then determined by reactivation and cross-linking experiments after dissociation in 0.1 M H3PO4 containing 1 M Na2SO4. The rate constant for the dimer association (which is the only rate-limiting step after acid dissociation) was k2 = (3.0 +/- 0.5) x 10(4) M-1 s-1. On the basis of the given two rate constants, the complete reassociation pattern of porcine lactic dehydrogenase after dissociation and denaturation in 6 M Gdn . HCl can be described by the kinetic model (formula: see text).     

Characterization of the folding and unfolding reactions of a small beta-barrel protein of novel topology, the MTCP1 oncogene product P13.

The equilibrium and kinetic folding properties of a small oncogene product, P13(MTCP1), of novel topology have been investigated using perturbation by guanidine hydrochloride and observation by fluorescence, circular dichroism and two-dimensional heteronuclear NMR spectroscopy. The structure of P13(MTCP1) is comprised of a canonical filled beta-barrel, although the topology of the structure is absolutely unique, rendering the folding properties of this protein of great interest. Equilibrium measurements of the intrinsic fluorescence emission spectrum, the fluorescence decay, the circular dichroism spectrum and the (15)N-(1)H heteronuclear single quantum coherence (HSQC) correlation spectrum as a function of increasing concentrations of denaturant showed no evidence for the population of any equilibrium intermediates, although negative amplitudes on the blue edge of the tryptophan emission and loss of intensity of the native HSQC correlation peaks were indicative of increased conformational dynamics at low denaturant concentrations. The free energy and cooperativity of unfolding as observed by fluorescence and circular dichroism were in relatively good agreement, also consistent with a two-state transition. Kinetics measurements of the fluorescence emission as a function of denaturant concentration revealed that P13(MTCP1) is the slowest folding beta-structure protein reported to date. Comparison of the activation cooperativity values (m(f) and m(u)) indicates that the structure of the transition state is quite close to the folded state in terms of exposed surface area. The calculated contact order of P13(MTCP1) is relatively low and does not appear to explain its slow rate of folding. We suggest that the complex topology of this protein, which would require the ordering of the beta-barrel through a long loop joining the two L-shaped components of the barrel, could provide an explanation for this slow folding. Copyright.     

Evidence for active intermediates during the reconstitution of yeast phosphoglycerate mutase

The reconstitution of the tetrameric phosphoglycerate mutase from bakers' yeast after denaturation in guanidine hydrochloride has been studied. When assays are performed in the presence of trypsin, it is found that reactivation parallels the regain of tetrameric structure. However, in the absence of trypsin, the regain of activity is more rapid, suggesting that monomeric and dimeric intermediates possess partial activity (35% of the value of native enzyme) which is sensitive to trypsin. When reconstitution is studied in the presence of substrates, it is again found that monomeric and dimeric intermediates possess 35% activity. Under these latter conditions, the activity of the monomer but not of the dimer is sensitive to trypsin.     

Effects of lactic acid and NaCl on creatine kinase from rabbit muscle.

The lactic acid induced unfolding and the salt-induced folding of creatine kinase (CK) were studied by enzyme activity, fluorescence emission spectra, circular dichroism spectra, and native polyacrylamide gel electrophoresis. The results showed that the kinetics of CK inactivation was a monophase process. Lactic acid caused inactivation and unfolding of CK with no aggregation during CK denaturation. The unfolding of the whole molecule and the inactivation of CK in solutions of different concentration of lactic acid were compared. Much lower lactic acid concentration values were required to bring about inactivation than were required to produce significant conformational changes of the enzyme molecule. At higher concentrations of lactic acid (more than 0.2 mM) the CK dimers were partially dissociated, as proved by native polyacrylamide gel electrophoresis. NaCl induced the molten globule state with a compact structure after CK was denatured with 0.8 mM lactic acid, and the increasing of anions led to a tight side-chain. The above results suggest that the effect of lactic acid differed from that of other denaturants such as guanidine hydrochloride, HCI, or urea during CK folding, and the molten globule state indicates that intermediates exist during CK folding.     

Artificial chaperone mediated refolding of xylanase from an alkalophilic thermophilic Bacillus sp. Implications for in vitro protein renaturation via a folding intermediate.

To gain insight into the molecular aspects of unfolding/refolding of enzymes from extremophilic organisms, we have used xylanase from an alkalophilic thermophilic Bacillus as the model system. Kinetics of denaturation/renaturation were monitored using intrinsic fluorescence studies. The protein fluorescence measurements suggested a putative intermediate state present in 0.08 M guanidine hydrochloride with an emission maximum of 345 nm; the far-UV circular dichroism spectra revealed content of secondary structure similar to the native enzyme. Studies with the fluorescent apolar probe 1-anilinonapthalene-8-sulfonate (1,8-ANS) were consistent with the presence of increased hydrophobic surfaces as compared with the native or fully unfolded protein. The refolding of Xyl II, was attempted by a relatively new strategy using an artificial chaperone assisted two-step method. The unfolded xylanase was found to bind to the detergent transiently and the subsequent addition of methyl-beta-cyclodextrin helped to strip the detergent and assist in the folding. Our findings suggested that the detergent stabilized a putative intermediate in the folding pathway seemingly equivalent to the folding state described as molten globule. The reactivation of Xyl II was affected by ionic as well as nonionic detergents. However, the cationic detergent cetyltrimethylammonium bromide (CTAB) provided a maximum reactivation (threefold) of the enzyme. The 'delayed detergent addition' experiments revealed that the detergent acts by suppressing the initial aggregate formation and not by dissolving aggregates. The relevance of our findings to the role of artificial chaperones in vivo is discussed.     

Solvent-tuning the collapse and helix formation time scales of lambda(6-85)*

The lambda(6-85)(*) pseudo-wild type of lambda repressor fragment is a fast two-state folder (k(f) approximately 35 microsec(-1) at 58 degrees C). Previously, highly stable lambda(6-85)(*) mutants with k(f) > 30 microsec(-1) have been engineered to fold nearly or fully downhill. Stabilization of the native state by solvent tuning might also tune lambda(6-85)(*) away from two-state folding. We test this prediction by examining the folding thermodynamics and kinetics of lambda(6-85)(*) in a stabilizing solvent, 45% by weight aqueous ethylene glycol at -28 degrees C. Detection of kinetics by circular dichroism at 222 nm (sensitive to helix content) and small angle X-ray scattering (measuring the radius of gyration) shows that refolding from guanidine hydrochloride denatured conditions exhibits very different time scales for collapse and secondary structure formation: the two processes become decoupled. Collapse remains a low-barrier activated process, while the fastest of several secondary structure formation time scales approaches the downhill folding limit. Two-state folding of lambda(6-85)(*) is not a robust process.     

Characterization of kinetic and thermodynamic phases in the prefolding process of bovine pancreatic ribonuclease A coupled with fast SS formation and SS reshuffling

In the redox-coupled oxidative folding of a protein having several SS bonds, two folding phases are usually observed, corresponding to SS formation (oxidation) with generation of weakly stabilized heterogeneous structures (a chain-entropy losing phase) and the subsequent intramolecular SS rearrangement to search for the native SS linkages (a conformational folding phase). By taking advantage of DHS(ox) as a highly strong and selective oxidant, the former SS formation phase was investigated in detail in the oxidative folding of RNase A. The folding intermediates obtained at 25 °C and pH 4.0 within 1 min (1S°-4S°) showed different profiles in the HPLC chromatograms from those of the intermediates obtained at pH 7.0 and 10.0 (1S-4S). However, upon prolonged incubation at pH 4.0 the profiles of 1S°-3S° transformed slowly to those similar to 1S-3S intermediate ensembles via intramolecular SS reshuffling, accompanying significant changes in the UV and fluorescence spectra but not in the CD spectrum. Similar conversion of the intermediates was observed by pH jump from 4.0 to 8.0, while the opposite conversion from 1S-4S was observed by addition of guanidine hydrochloride to the folding solution at pH 8.0. The results demonstrated that the preconformational folding phase coupled with SS formation can be divided into two distinct subphases, a kinetic (or stochastic) SS formation phase and a thermodynamic SS reshuffling phase. The transition from kinetically formed to thermodynamically stabilized SS intermediates would be induced by hydrophobic nucleation as well as generation of the native interactions.     

The nature of the rate-limiting steps in the refolding of the cofactor-dependent protein aspartate aminotransferase

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 m guanidine hydrochloride for different periods of time. Whereas reactivation of equilibrium-unfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k approximately 0.75 min(-1) at 20 degrees C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stopped-flow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5'-phosphate (PLP) to the polypeptide is much slower (k approximately 0.08 min(-1)). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.     

根霉葡萄糖淀粉酶的复性复活动力学研究

本文利用荧光、紫外差光谱研究了根霉葡萄糖淀粉酶在盐酸胍变性后的复性、复活动力学。结果表明,该酶在小于4mol/L盐酸胍中变性是可逆的,其复性过程遵循一级反应方程。酶复活过程是由两个一级反应组成的复合反应,构象变化速度与复活过程中较快的反应速度相差无几,这可能是在Trp及Tyr微区的构象变化基本完成之后,酶活力恢复还没有完成造成的。     

Investigation of the folding pathway of the TEM-1 β-lactamase

The TEM-1 β-lactamase is a globular protein containing 12 proline residues. The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism. The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state. Thisstate is 5.2 ± 0.4 kcal/mol less stable than the native conformation and 5.7 ± 0.2 kcal/mol more stable than the fully denaturedprotein. This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the “molten globule” is discussed. Refolding kinetic experimentsrevealed the existence of a transient intermediate conformation between thethermodynamically stable intermediate and the native protein. This transient intermediate appears rapidly during the folding reaction. It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation. The final refolding step of the TEM-1 β-lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probablyreflect a very complex process kinetically limited by proline cis/transisomerization. © 1995 Wiley-Liss, Inc.