Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.     

Unfolding of Plasmodium falciparum triosephosphate isomerase in urea and guanidinium chloride: evidence for a novel disulfide exchange reaction in a covalently cross-linked mutant

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.     

Guanidine-induced denaturation of beta-glycosidase from Sulfolobus solfataricus expressed in Escherichia coli

Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL-1. Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1. Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.     

Chemical denaturation: potential impact of undetected intermediates in the free energy of unfolding and m-values obtained from a two-state assumption

The chemical unfolding transition of a protein was simulated, including the presence of an intermediate (I) in equilibrium with the native (N) and unfolded (U) states. The calculations included free energies of unfolding, DeltaGuw, in the range of 1.4 kcal/mol to 10 kcal/mol and three different global m-values. The simulations included a broad range of equilibrium constants for the N left arrow over right arrow I process. The dependence of the N <--> I equilibrium on the concentration of denaturant was also included in the simulations. Apparent DeltaGuw and m-values were obtained from the simulated unfolding transitions by fitting the data to a two-state unfolding process. The potential errors were calculated for two typical experimental situations: 1) the unfolding is monitored by a physical property that does not distinguish between native and intermediate states (case I), and 2) the physical property does not distinguish between intermediate and unfolded states (case II). The results obtained indicated that in the presence of an intermediate, and in both experimental situations, the free energy of unfolding and the m-values could be largely underestimated. The errors in DeltaGuw and m-values do not depend on the m-values that characterize the global N <--> U transition. They are dependent on the equilibrium constant for the N <--> I transition and its characteristic m1-value. The extent of the underestimation increases for higher energies of unfolding. Including no random error in the simulations, it was estimated that the underestimation in DeltaGuw could range between 25% and 35% for unfolding transitions of 3-10 kcal/mol (case I). In case II, the underestimation in DeltaGuw could be even larger than in case I. In the same energy range, a 50% error in the m-value could also take place. The fact that most of the mutant proteins are characterized by both a lower m-value and a lower stability than the wild-type protein suggests that in some cases the results could have been underestimated due to the application of the two-state assumption.     

Unfolding of the tetrameric loop deletion mutant of ROP protein is a second-order reaction

Comprehensive kinetic studies were carried out on the unfolding properties of RM6 as a function of GdnHCl concentration and temperature. This protein is a mutant resulting from the dimeric wild-type CoLE1-ROP protein by deletion of 5 amino acids (Asp 30, Ala 31, Asp 32, Glu 33, Gln 34) in the loop of each monomer. The deletion has dramatic consequences. The dimeric 4-alpha-helix structure characteristic of the wild-type protein is completely reorganized and the RM6 structure can be described as a tetrameric alpha helix of extended monomers without loops. These extraordinary structural changes are accompanied by an enormous increase in transition temperature from 71 to 101 degreesC. These features have been discussed in a separate publication (1). The remarkable change in thermal stability of RM6 should be reflected in significant changes in the folding rate constants. This was observed in the present unfolding studies. Decay of tetrameric RM6 was monitored by circular dichroism (CD) and fluorescence to probe for changes in both secondary and tertiary structure, respectively. The identity of the kinetic parameters obtained from the two techniques supports the view that secondary and tertiary structure break down simultaneously. However, the most intriguing result is the finding that unfolding of tetrameric RM6 can be described very well by a second-order reaction. The magnitude of the second-order rate constant k2 varies dramatically with both temperature and denaturant concentration. At 25 degreesC and 6.5 M GdnHCl concentration k2 is 4200 L.(mol of dimer)-1.s-1, whereas at 4.4 M GdnHCl a value of k2 = 0.9 L.(mol of dimer)-1.s-1 is observed. Correspondingly, apparent activation enthalpies show a strong increase from DeltaH# = 29.1 kJ.mol-1 at 6. 5 M GdnHCl to Delta H# = 79.7 kJ.mol-1 at 4.4 M GdnHCl. A mechanism involving a dimeric intermediate is suggested which permits a consistent interpretation of the findings.     

Conversion of native oligomeric to a modified monomeric form of human C-reactive protein

C-reactive protein (CRP) is a pentameric oligoprotein composed of identical 23 kD subunits which can be modified by urea-chelation treatment to a form resembling the free subunit termed modified CRP (mCRP). mCRP has distinct physicochemical, antigenic, and biologic activities compared to CRP. The conditions under which CRP is converted to mCRP, and the molecular forms in the transition, are important to better understand the distinct properties of mCRP and to determine if the subunit form can convert back to the pentameric native CRP form. This study characterized the antigenic and conformational changes associated with the interconversion of CRP and mCRP. The rate of dissociation of CRP protomers into individual subunits by treatment in 8 M urea-10 mM EDTA solution was rapid and complete in 2 min as assayed by an enzyme-linked immunofiltration assay using monoclonal antibodies specific to the mCRP. Attempts to reconstitute pentameric CRP from mCRP under renaturation conditions were unsuccessful, resulting in a protein retaining exclusively mCRP characteristics. Using two-dimensional urea gradient gel electrophoresis, partial rapid unfolding of the pentamer occurred above 3 M urea, a subunit dissociation at 6 M urea, and further subunit unfolding at 6-8 M urea concentrations. The urea gradient electrophoresis results suggest that there are only two predominant conformational states occurring at each urea transition concentration. Using the same urea gradient electrophoresis conditions mCRP migrated as a single molecular form at all urea concentrations showing no evidence for reassociation to pentameric CRP or other aggregate form. The results of this study show a molecular conversion for an oligomeric protein (CRP) to monomeric subunits (mCRP) having rapid forward transition kinetics in 8 M urea plus chelator with negligible reversibility.     

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain (residues 1-319), which is dimeric and forms tyrosyladenylate, and a C-terminal domain (residues 320-419), which binds the anticodon arm of tRNATyr. The N-terminal domain has the characteristic fold of the class I aminoacyl-tRNA synthetases. The unfolding of the N-terminal domain by urea at 25 degreesC under equilibrium conditions was monitored by its intensities of light emission at 330 and 350 nm, the ratio of these intensities, its ellipticity at 229 nm, and its partition coefficient, in spectrofluorometry, circular dichroism, and size-exclusion chromatography experiments, respectively. These experiments showed the existence of an equilibrium between the native dimeric state of the N-terminal domain, a monomeric intermediate state, and the unfolded state. The intermediate was compact and had secondary structure, and its tryptophan residues were partially buried. These properties of the intermediate and its inability to bind 1-anilino-8-naphthalenesulfonate showed that it was not in a molten globular state. The variation of free energy deltaG(H2O) and its coefficient m of dependence on the concentration of urea were, respectively, 13.8 +/- 0.2 kcal.mol-1 and 0.9 +/- 0.1 kcal.mol-1.M-1 for the dissociation of the native dimer and 13.9 +/- 0.6 kcal.mol-1 and 2.5 +/- 0.1 kcal.mol-1.M-1 for the unfolding of the monomeric intermediate.     

Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain

The equilibrium unfolding reaction of the C-terminal 80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, delta GH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 x 10(-8) M. Equilibrium sedimentation experiments give a Kd of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.     

Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor

The urea-induced equilibrium unfolding of the Escherichia coli Trp repressor (TR) is a two-state process, involving the native dimeric and unfolded monomeric species. Kinetic studies, however, reveal the presence of transient intermediates that appear only during the folding of the 107-residue protein [Gittelman, M. G., & Matthews, C. R. (1990) Biochemistry 29, 7011-7020]. In order to gain insight into the complex kinetic folding mechanism, the sequence of TR was reduced to the amino-terminal 66 residues, corresponding to the dimerization domain. Two polypeptides, 2-66 and NHis-7-66, were shown to be dimeric at 25 degrees C by size exclusion chromatography and to retain native-like spectroscopic features as evidenced by near- and far-UV circular dichroism and fluorescence spectroscopy. The equilibrium properties of the urea-induced folding of these core fragments were examined by intrinsic tryptophan fluorescence and circular dichroism and found to be well described by a two-state model. At 25 degrees C, the stabilities of both fragments are 14 kcal mol(-1), as compared to the 24 kcal mol(-1) observed for full-length TR. In contrast, the thermal denaturation of [2-66]2 and full-length TR are three-state processes; the midpoint of the transition monitored by absorbance at 292 nm precedes that monitored by circular dichroism at 222 nm. Global analysis of the thermal data as a function of monomer concentration suggests that both the full-length and [2-66]2 TR variants unfold via a dimeric intermediate. Taken together, these results demonstrate that the [2-66]2 fragment constitutes a well-structured, independently folding subdomain of TR that may be useful in elucidating the properties of the transient intermediates observed in the folding of the full-length protein. The dimeric intermediate observed in the thermal denaturation of [2-66]2 suggests that it may be possible to further reduce the core sequence while maintaining the ability to dimerize.     

The barriers in the bimolecular and unimolecular folding reactions of the dimeric core domain of Escherichia coli Trp repressor are dominated by enthalpic contributions

The kinetic folding mechanism of the isolated dimerization domain of E. coli Trp repressor, [2-66]2 TR, consists of a nearly diffusion-limited association reaction to form a dimeric intermediate, I2, which is then converted to the native, folded dimeric species, N2 by a first-order folding step (preceding paper in this issue). The two transition states traversed in the folding of [2-66]2 TR were characterized by monitoring the folding and unfolding reactions by stopped-flow fluorescence as a function of temperature and urea. For both transition states, the barriers are dominated by the enthalpic component; the entropic component accelerates the association reaction but has little effect on the subsequent rearrangement reaction. The transition state between I2 and N2 is relatively nativelike, as determined by the sensitivity of the rate constants to denaturant. This study also highlights the key role of solvent entropy in determining the magnitude of the relative free energy of the transition states and the ground states. The positive entropy change for the I2 to N2 reaction, presumably arising from the release of solvent from hydrophobic surfaces, is the driving force for this final folding step, offsetting an unfavorable enthalpic term.     

TCI temperamental scores in bulimia nervosa patients and normal women with and without diet experiences

The tailspike protein (TSP) of bacteriophage P22 is a homotrimeric multifunctional protein responsible for cell attachment and hydrolysis of the Salmonella typhimurium host cell receptor. Despite the folding of TSP involves the formation of thermolabile intermediates, the mature protein is extremely resistant to heat and detergent denaturation. We have analyzed the thermal resistance and unfolding pathway of two mutant, functional TSPs carrying end-terminal peptide fusions. Whereas the C-terminal fusion has minor effects on the TSP stability, the presence of a 23-mer foreign peptide at the N terminus (protein ATSP) results in a significant enhancement of the thermal resistance by retarding the first transition step of the unfolding process. At 65 degrees C and in 2% SDS, the unfolding rate constant for the transition from the native to the unfolding intermediate is 9.3 x 10(-4) s(-1) for ATSP versus 1.7 x 10(-3) s(-1) for wild-type TSP. On the other hand, the electrophoretic mobility of ATSP intermediates is greatly affected, proving structural modifications induced by the fused peptide. These results suggest a critical participation of the N-terminal domain in the unfolding kinetic barriers generated during the TSP denaturation pathway.     

Thermodynamic characterization of the conformational stability of the homodimeric protein, pea lectin

The conformational stability of the homodimeric pea lectin was determined by both isothermal urea-induced and thermal denaturation in the absence and presence of urea. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with the unfolding of the protein. The data not only conform to the simple A2 if 2U model of unfolding but also are well described by the linear extrapolation model for the nature of denaturant-protein interactions. In addition, both the conformational stability (DeltaGs) and the DeltaCp for the protein unfolding is quite high, at about 18.79 kcal/mol and 5.32 kcal/(mol K), respectively, which may be a reflection of the relatively larger size of the dimeric molecule (Mr 49 000) and, perhaps, a consequent larger buried hydrophobic core in the folded protein. The simple two-state (A2 if 2U) nature of the unfolding process, with the absence of any monomeric intermediate, suggests that the quaternary interactions alone may contribute significantly to the conformational stability of the oligomer-a point that may be general to many oligomeric proteins.     

Characterization of a subunit structure and stability of the recombinant porin from Neisseria gonorrhoeae

An outer membrane PIA protein from Neisseria gonorrhoeae strain FA19 was expressed in Escherichia coli and refolded in vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50 degrees C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80 degrees C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind with Kd=80 and 130 microM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.     

Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species

The kinetics of refolding of guanidinium chloride-denatured rabbit MM-creatine kinase was investigated. Recovery of enzymatic activity is biphasic, depending on the temperature but not on the protein or DTT concentration. Only 45% of the original, active dimeric form is recovered even after several hours of refolding. The reactivation yield is limited by the accumulation of a highly stable but nonproductive monomeric species. The ratio of "correct" to "incorrect" forms depends on the duration of exposure to the denaturant, which may be consistent with the existence of a heterogeneous population of unfolded states with regard to proline isomerization. The first fast reaction observed during renaturation results in the appearance of collapsed monomeric states, displaying features of a pre-molten globule state. These burst species are rapidly transformed into more structured monomers resembling a molten globule state possessing a partially folded C-terminal domain. A proportion of these latter transient intermediates (45%) associates into an active dimer, while the remainder (55%) is trapped by reshuffling in a monomeric dead-end product. Our results strongly indicate that (i) the dimeric state is a prerequisite for the expression of catalytic activity, (ii) the kinetic intermediates of refolding are very similar to those observed during equilibrium unfolding, and (iii) refolding of creatine kinase in these conditions is limited by the accumulation of inactive misfolded nondimerizable monomer.     

Kinetics and energetics of subunit dissociation/unfolding of TIM: the importance of oligomerization for conformational persistence and chemical stability of proteins

Kinetics of unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM) were measured as a function of guanidine hydrochloride (GdnHCl) concentration. From the rate constants of these processes, the activation free-energy barriers (delta G++) were calculated using the Arrhenius equation. Assuming a linear dependence of delta G++ on the concentration of GdnHCl, activation energies in the absence of GdnHCl were estimated. The Gibbs free-energy change of dissociation/unfolding (delta G) was determined from GdnHCl unfolding curves in equilibrium. Using these data and the literature value for the bimolecular association rate constant of folded TIM monomers [Zabori, S., Rudolph, R., and Jaenicke, R. (1980) Z. Naturforsch. 35C, 999-1004], a model was developed that fully describes both kinetics and energetics of subunit dissociation/unfolding of TIM. Unfolded TIM monomers are susceptible to proteolytic digestion and thiol oxidation, while native TIM is resistant to both. The present model explains how the dimeric nature of TIM decreases the frequency of subunit unfolding by several orders of magnitude, thus increasing the chemical stability of the protein. Furthermore, the model also explains the recently demonstrated persistence (on a time scale of hours to days) of conformational heterogeneity of native TIM dimers [Rietveld, A. W. M., and Ferreira, S. T. (1996) Biochemistry 35, 7743-7751]. Again, it appears that the dimeric nature of TIM is essential for this behavior.     

Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule

The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.     

A partially folded intermediate during tubulin unfolding: its detection and spectroscopic characterization

The unfolding reaction of the dimeric protein tubulin, isolated from goat brain, was studied using fluorescence and circular dichroism techniques. The unfolding of the tubulin dimer was found to be a two-step process at pH 7. The first step leads to the formation of an intermediate conformation, stable at around 1-2 M urea, followed by a second step that was due to unfolding of the intermediate state. At pH 3, the urea-induced biphasic unfolding profiles obtained at pH 7 became a one-step process indicating that a stable intermediate was also formed at this pH. The intermediate at pH 3 was more stable toward urea denaturation than that at pH 7. The intermediate state has about 60% secondary structure, partially exposed aromatic residues, and less tertiary structure as compared to the native states. Also, hydrophobic surfaces were more exposed in the intermediate than in the native or unfolded states. These results indicate that the intermediate state observed during tubulin unfolding is not only distinct from both the native and unfolded forms but also possesses some properties characteristic of a molten globule.     

Mutagenic and thermodynamic analyses of residual structure in the alpha subunit of tryptophan synthase

The alpha subunit of tryptophan synthase from Escherichia coli has been previously shown to contain residual structure at 5 M urea, conditions where the secondary structure is entirely disrupted and the tyrosine residues are exposed to solvent [Saab-Rincón, G., Froebe, C. L., & Matthews, C. R. (1993) Biochemistry 32, 13981-13990]. The residual structure can be monitored by one-dimensional NMR spectroscopy studies of histidine 92 whose C epsilon proton is sensitive to the slow exchange between this form and the unfolded protein. The temperature dependence of the cooperative urea-induced unfolding transition between intermediate and unfolded forms demonstrates that this process involves negative values for both the enthalpy and entropy changes at 25 degrees C. The effects of replacements of several nonpolar side chains adjacent to histidine 92 on the slopes and midpoints of the unfolding transition curve show that these side chains participate in the residual structure. A 15-residue peptide spanning histidine 92 and the mutated residues, however, is not sufficient to define this structure. These results demonstrate that the residual structure in the alpha subunit is stabilized by the hydrophobic effect and may involve side chains which are distant in sequence to histidine 92.     

Active monomeric and dimeric forms of Pseudomonas putida glyoxalase I: evidence for 3D domain swapping

3D domain swapping of proteins involves the interconversion of a monomer containing a single domain-domain interface and a 2-fold symmetrical dimer containing two equivalent intermolecular interfaces. Human glyoxalase I has the structure of a domain-swapped dimer [Cameron, A. D., Olin, B., Ridderstr?m, M., Mannervik, B., and Jones, T. A. (1997) EMBO J. 16, 3386-3395] but Pseudomonas putida glyoxalase I has been reported to be monomeric [Rhee, H.-I., Murata, K., and Kimura, A. (1986) Biochem. Biophys. Res. Commun. 141, 993-999]. We show here that recombinant P. putida glyoxalase I is an active dimer (kcat approximately 500 +/- 100 s-1; KM approximately 0.4 +/- 0.2 mM) with two zinc ions per dimer. The zinc is required for structure and function. However, treatment of the dimer with glutathione yields an active monomer (kcat approximately 115 +/- 40 s-1; KM approximately 1.4 +/- 0.4 mM) containing a single zinc ion. The monomer is metastable and slowly reverts to the active dimer in the absence of glutathione. Thus, glyoxalase I appears to be a novel example of a single protein able to exist in two alternative domain-swapped forms. It is unique among domain-swapped proteins in that the active site and an essential metal binding site are apparently disassembled and reassembled by the process of domain swapping. Furthermore, it is the only example to date in which 3D domain swapping can be regulated by a small organic ligand.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.