Equilibrium unfolding of dimeric human prostatic acid phosphatase involves an inactive monomeric intermediate

Guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular weight, was investigated with activity measurements, size exclusion HPLC, tryptophan fluorescence, 1-anilinonaphtalene-8-sulfonate (ANS) binding and reactivity with 2-(4'-maleimidoanilino)naphthalene-6-sulfonate (MIANS). Equilibrium analysis was performed to shed light on the role of dimerization in the folding and stability of the catalytically active oligomeric protein. Unfolding was reversible, as verified by activity measurements and tryptophan fluorescence. The noncoincidence of the unfolding curves obtained by different techniques suggests the occurrence of a multiphasic process.The reaction of hPAP inactivation is accompanied by dissociation of the dimer into two monomers. The midpoint of this transition is at 0.65 M GdnHCl with 4.24+/-0.12 kcalmol(-1) free energy change. Binding of ANS to the inactive phosphatase monomer, especially remarkable in the region from 0.8 to 1.25M GdnHCl, suggests that the hydrophobic probe indicates exposition of the intersubunit hydrophobic surface and a loosening of the monomer's tertiary structure. Strong fluorescence of thiol group derivatives, the products of their reaction with MIANS, appears in a limited range of GdnHCl concentrations (1.2-1.6M). This shows that in the relaxed structure of the intermediate, the reagent is allowed to penetrate into the hydrophobic environment of the partially hidden thiol groups.The equilibrium unfolding reaction of hPAP, as monitored by tryptophan fluorescence, does not depend on the protein concentration and displays a single transition curve with a midpoint at 1.7 M GdnHCl and value of DeltaG(unf)(H(2)O)=3.38+/-0.08 kcalmol(-1) per monomer, a result implying that this transition is related to the conformational change of the earlier dissociated and already inactive subunit of the protein.     

Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.     

Thermal unfolding of monomeric and dimeric beta-lactoglobulins.

The thermal stabilities of dimeric bovine beta-lactoglobulin and monomeric equine beta-lactoglobulin were investigated at neutral pH by means of differential scanning calorimetry, circular dichroism, tryptophan fluorescence, and by binding of an hydrophobic probe. Differential scanning calorimetry showed the presence of two structural domains with different thermal stabilities in both proteins. Thermodynamic analysis of the calorimetric signal revealed that the two domains unfold independently according to a mechanism where an equilibrium step is followed by an irreversible transition. The spectroscopic data supported this model and allowed recognition of the structural regions corresponding to the more thermally stable domain. The differences in thermal stability between the two proteins can be primarily ascribed to the properties of the less stable domain.     

Equilibrium unfolding of an oligomeric protein involves formation of a multimeric intermediate state(s)

Superoxide dismutases (SODs) are important metalloenzymes which protect cells against oxidative stress by scavenging reactive superoxides. Missense mutations in SODs are known to lead to some familial cases of amyotrophic lateral sclerosis and several forms of cancers. In the present study, we investigate the guanidinium hydrochloride (GdnHCl)-induced equilibrium unfolding of apo-manganese superoxide dismutase (apo-MnSOD) isolated from Vibrio alginolyticus using a variety of biophysical techniques. GdnHCl-induced equilibrium unfolding of apo-MnSOD is non-cooperative and involves the accumulation of stable intermediate state(s). Results of 1-anilino-8-naphthalene sulfonate binding experiments suggest that the equilibrium intermediate state(s) accumulates maximally in 1.5 M GdnHCl. The intermediate state(s) appears to be obligatory and occurs both in the unfolding and refolding pathways. Size-exclusion chromatography and sedimentation velocity data reveal that the equilibrium intermediate state(s) is multimeric. To our knowledge, this is the first report of the identification of a multimeric intermediate in the unfolding pathway(s) of oligomeric proteins. The formation and dissociation of the multimeric intermediate state(s) appears to dictate the fate of the protein either to refold to its native conformation or misfold and form aggregates as observed in amyotrophic lateral sclerosis.     

Equilibrium unfolding of an oligomeric protein involves formation of a multimeric intermediate state(s)

Superoxide dismutases (SODs) are important metalloenzymes which protect cells against oxidative stress by scavenging reactive superoxides. Missense mutations in SODs are known to lead to some familial cases of amyotrophic lateral sclerosis and several forms of cancers. In the present study, we investigate the guanidinium hydrochloride (GdnHCl)-induced equilibrium unfolding of apo-manganese superoxide dismutase (apo-MnSOD) isolated from Vibrio alginolyticus using a variety of biophysical techniques. GdnHCl-induced equilibrium unfolding of apo-MnSOD is non-cooperative and involves the accumulation of stable intermediate state(s). Results of 1-anilino-8-naphthalene sulfonate binding experiments suggest that the equilibrium intermediate state(s) accumulates maximally in 1.5M GdnHCl. The intermediate state(s) appears to be obligatory and occurs both in the unfolding and refolding pathways. Size-exclusion chromatography and sedimentation velocity data reveal that the equilibrium intermediate state(s) is multimeric. To our knowledge, this is the first report of the identification of a multimeric intermediate in the unfolding pathway(s) of oligomeric proteins. The formation and dissociation of the multimeric intermediate state(s) appears to dictate the fate of the protein either to refold to its native conformation or misfold and form aggregates as observed in amyotrophic lateral sclerosis.     

Reversible equilibrium unfolding of triosephosphate isomerase from Trypanosoma cruzi in guanidinium hydrochloride involves stable dimeric and monomeric intermediates

The reversible guanidinium hydrochloride-induced unfolding of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was characterized under equilibrium conditions. The catalytic activity was followed as a native homodimeric functional probe. Circular dichroism, intrinsic fluorescence, and size-exclusion chromatography were used as secondary, tertiary, and quaternary structural probes, respectively. The change in ANS fluorescence intensity with increasing denaturant concentrations was also determined. The results show that two stable intermediates exist in the transition from the homodimeric native enzyme to the unfolded monomers: one (N(2*)) is a slightly more expanded, non-native, and active dimer, and the other is a partially expanded monomer (M) that binds ANS. Spectroscopic and activity data were used to reach a thermodynamic characterization. The results indicate that the Gibbs free energies for the partial reactions are 4.5 (N(2) <==> N(2*)), 65.8 (N(2*) <==> 2M), and 17.8 kJ/mol (M <==> U). It appears that TcTIM monomers are more stable than those found for other TIM species (except yeast TIM), where monomer stability is only marginal. These results are compared with those for the guanidinium hydrochloride-induced denaturation of TIM from different species, where despite the functional and three-dimensional similarities, a remarkable heterogeneity exists in the unfolding pathways.     

Zinc induces unfolding and aggregation of dimeric arginine kinase by trapping reversible unfolding intermediate

Arginine kinase plays an important role in the cellular energy metabolism of invertebrates. Dimeric arginine kinase (dAK) is unique in some marine invertebrates. The effects of Zn2(+) on the unfolding and aggregation of dAK from the sea cucumber Stichopus japonicus were investigated. Our results indicated that Zn2(+) caused dAK inactivation accompanied by conformational unfolding, the exposure of hydrophobic surface, and aggregation. Kinetic studies showed the inactivation and unfolding of dAK followed biphasic kinetic courses. Zn2(+) can affect unfolding and refolding of dAK by trapping the reversible intermediate. Our study provides important information regarding the effect of Zn2(+) on metabolic enzymes in marine invertebrates.     

Evidence for a monomeric intermediate in the reversible unfolding of F factor TraM

F factor TraM is essential for efficient bacterial conjugation, but its molecular function is not clear. Because the physical properties of TraM may provide clues to its role in conjugation, we have characterized the TraM oligomerization equilibrium. We show that the reversible unfolding transition is non-two-state, indicating the presence of at least one intermediate. Analytical ultracentrifugation experiments indicate that the first phase of unfolding involves dissociation of the tetramer into folded monomers, which are subsequently unfolded to the denatured state in the second phase. Furthermore, we show that a C-terminal domain isolated by limited proteolysis is tetrameric in solution, like the full-length protein, and that its loss of structure correlates with dissociation of the TraM tetramer. Unfolding of the individual domains indicates that the N- and C-terminal regions act cooperatively to stabilize the full-length protein. Together, these experiments suggest structural overlap of regions important for oligomerization and DNA binding. We propose that modulating the oligomerization equilibrium of TraM may regulate its essential activity in bacterial conjugation.     

Reversible dissociation and unfolding of aspartate aminotransferase from Escherichia coli: characterization of a monomeric intermediate

The unfolding and dissociation of the dimeric enzyme aspartate aminotransferase (D) from Escherichia coli by guanidine hydrochloride have been investigated at equilibrium. The overall process was reversible, as judged from almost complete recovery of enzymic activity after dialysis of 0.7 mg of denatured protein/mL against buffer. Unfolding and dissociation were monitored by circular dichroism and fluorescence spectroscopy and occurred in three separate phases: D in equilibrium 2M in equilibrium 2M* in equilibrium 2U. The first transition at about 0.5 M guanidine hydrochloride coincided with loss of enzyme activity. It was displaced toward higher denaturant concentrations by the presence of either pyridoxal 5'-phosphate or pyridoxamine 5'-phosphate and toward lower denaturant concentrations by decreasing the protein concentration. Therefore, bound coenzyme stabilizes the dimeric state, and the monomer (M) is inactive because the shared active sites are destroyed by dissociation of the dimer. M was converted to M* and then to the fully unfolded monomer (U) in two subsequent transitions. M* was stable between 0.9 and 1.1 M guanidine hydrochloride and had the hydrodynamic radius, circular dichroism, and fluorescence of a monomeric, compact "molten globule" state.     

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.     

Characterization of a dimeric unfolding intermediate of bovine serum albumin under mildly acidic condition

Protein aggregation is a well-known phenomenon related to serious medical implications. Bovine serum albumin (BSA), a structural analogue of human serum albumin, has a natural tendency for aggregation under stress conditions. While following effect of moderately acidic pH on BSA, a state was identified at pH 4.2 having increased light scattering capability at 350 nm. It was essentially a dimer devoid of disulphide linked large aggregates as observed from 'spin column' experiments, gel electrophoresis and ultra-centrifugations. Its surface hydrophobic character was comparable to the native conformer at pH 7.0 as observed by the extraneous fluorescence probes pyrene and pyrene maleimide but its interactions with 1-anilino 8-naphthelene sulphonic acid was more favorable. Dimerization was irreversible between pH 4.2 and 7.0 even after treatment with DTT. The role of the only cysteine-34 residue was investigated where modification with reagents of arm length bigger than 6 A prevented dimerization. Molecular modeling of BSA indicated that cys-34 resides in a cleft of 6 A depth. This indicated that the area surrounding the cleft plays important role in inducing the dimerization.     

Analysis of betaB1-crystallin unfolding equilibrium by spin and fluorescence labeling: evidence of a dimeric intermediate

A central step in understanding lens aging is to characterize the thermodynamic stability of its proteins and determine the consequences of changes in the primary sequence on their folding equilibria. For this purpose, destabilized mutations were introduced in betaB1-crystallin targeting the domain interface within the fold of a subunit. Global unfolding was monitored by tryptophan fluorescence while concomitant structural changes at the dimer interface were monitored by fluorescence and spin labels. Both spectral probes report explicit evidence of multi-state unfolding equilibrium. The biphasic nature of the unfolding curves was more pronounced at higher protein concentration. Distinct shifts in the midpoint of the second transition reflect the population of a dimeric intermediate. This intermediate may be a critical determinant for the life-long stability of the beta-crystallins and has important consequences on interactions with alpha-crystallin.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure

The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUalpha(2) and EcHUbeta(2), and a heterodimer, EcHUalphabeta. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E.coli HU dimers and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N(2)<-->2D), this present thermodynamic study shows that the three E.coli dimers melt according to a two-step mechanism (N(2)<-->I(2)<-->2D). The native dimer, N(2), melts partially into a dimeric intermediate, I(2), which in turn yields the unfolded monomers, D. In addition, the crystal structure of the EcHUalpha(2) dimer has been solved. Comparative thermodynamic and structural analysis between EcHUalpha(2) and the HU homodimer from Bacillus stearothermophilus suggests that the E.coli dimer is constituted by two subdomains of different energetic properties. The CD study indicates that the intermediate, I(2), corresponds to an HU dimer having partly lost its alpha-helices. The partially unfolded dimer I(2) is unable to complex with high-affinity, single-stranded break-containing DNA. These structural, thermodynamic and functional results suggest that the N(2)<-->I(2) equilibrium plays a central role in the physiology of E.coli HU. The I(2) molecular species seems to be the EcHUbeta(2) preferential conformation, possibly related to its role in the E.coli cold-shock adaptation. Besides, I(2) might be required in E.coli for the HU chain exchange, which allows the heterodimer formation from homodimers.     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.     

A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2. 2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23. 5 A). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.     

Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates

Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has α-helix and β-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.     

Thermal inactivation and unfolding of a dimeric arginine kinase

Thermal inactivation and unfolding of the dimeric arginine kinase (AK) from sea cucumber Stichopus japonicus was investigated. The activation energy was calculated to be 388 kJ/mol. Based on the analysis of the denaturation course at 58 degrees C, a model is suggested for the thermal unfolding of this dimeric AK. In addition, the effect of free Mg(2+) and the potential biological significance on the thermal unfolding of dimeric AK is discussed.     

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.