Unexpected diversity of chlorite dismutases: a catalytically efficient dimeric enzyme from Nitrobacter winogradskyi

Chlorite dismutase (Cld) is a unique heme enzyme catalyzing the conversion of ClO(2)(-) to Cl(-) and O(2). Cld is usually found in perchlorate- or chlorate-reducing bacteria but was also recently identified in a nitrite-oxidizing bacterium of the genus Nitrospira. Here we characterized a novel Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. Its three-dimensional (3D) crystal structure revealed a dimer of two identical subunits, which sharply contrasts with the penta- or hexameric structures of other chlorite dismutases. Despite a truncated N-terminal domain in each subunit, this novel enzyme turned out to be a highly efficient chlorite dismutase (K(m) = 90 μM; k(cat) = 190 s(-1); k(cat)/K(m) = 2.1 × 10(6) M(-1) s(-1)), demonstrating a greater structural and phylogenetic diversity of these enzymes than was previously known. Based on comparative analyses of Cld sequences and 3D structures, signature amino acid residues that can be employed to assess whether uncharacterized Cld-like proteins may have a high chlorite-dismutating activity were identified. Interestingly, proteins that contain all these signatures and are phylogenetically closely related to the novel-type Cld of N. winogradskyi exist in a large number of other microbes, including other nitrite oxidizers.     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

Marburg virus glycoprotein GP2: pH-dependent stability of the ectodomain α-helical bundle

Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV requires fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein, GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔG(unf,H(2)O), of 33.4 ± 2.5 kcal/mol and melting temperature, T(m), of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH, with higher stability under lower-pH conditions (T(m) values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. On the basis of these results, we hypothesize that the pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism for controlling conformational preferences such that the six-helix bundle "postfusion" state is preferred under conditions of appropriately matured endosomes.     

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.     

Monomeric Banana Lectin at Acidic pH Overrules Conformational Stability of Its Native Dimeric Form

Banana lectin (BL) is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS) binding, size exclusion chromatography (SEC) and dynamic light scattering (DLS). During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml) at pH 2.0 while single peak (61.45 ml) at pH 7.4. The hydrodynamic radii (R _h) of native BL was 2.9 nm while at pH 2.0 two species were found with R _h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high C_m and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.     

Multiple unfolding states of glutathione transferase from Physa acuta (Gastropoda [correction of Gastropada]: Physidae)

The equilibrium unfolding of the major Physa acuta glutathione transferase isoenzyme (P. acuta GST(3)) has been performed using guanidinium chloride (GdmCl), urea, and acid denaturation to investigate the unfolding intermediates. Protein transitions were monitored by intrinsic fluorescence. The results indicate that unfolding of P. acuta GST(3) using GdmCl (0-3.0M) is a multistep process, i.e., three intermediates coexist in equilibrium. The first intermediate, a partially dissociated dimer, exists at low GdmCl concentration (approximately at 0.7M). At 1.2M GdmCl, a dimeric intermediate with a compact structure was observed. This intermediate undergoes dissociation into structural monomers at 1.75M of GdmCl. The monomeric intermediate started to be completely unfolding at higher GdmCl concentrations (>1.8M). Unfolding using urea (0-7.0M) and acid-induced structures as well as the fluorescence of 8-anilino-1-naphthalenesulfonate in the presence of different GdmCl concentrations confirmed that the unfolding is a multistep process. At concentrations of GdmCl or urea less than the midpoints or at the midpoint pH (pH 4.2-4.6), the unfolding transition is protein concentration independent and involved a change in the subunit tertiary structure yielding a partially active dimeric intermediate. The binding of glutathione to the enzyme active site stabilizes the native dimeric state.     

Thermal aggregation of ribonuclease A. A contribution to the understanding of the role of 3D domain swapping in protein aggregation

By lyophilizing RNase A from 40% acetic acid solutions, two dimeric aggregates, the "minor" and "major" dimers (named here N-dimer and C-dimer, respectively), form by 3D domain swapping at a ratio of 1:4. Trimeric and tetrameric aggregates are also obtained. The two dimers and the higher oligomers also form without a lyophilization step. By keeping RNase A dissolved at a high concentration (generally 200 mg/ml) in various media at temperatures ranging from 23 to 70 degrees C for times varying from a few minutes to 2 h, various oligomers, in particular the two dimeric conformers, formed in quite different amounts, often inverting their relative quantities depending on the more or less severe unfolding conditions. When unfolding mainly concerned the N terminus of the protein, richer in hydrophilic residues, the N-dimer, formed by 3D domain swapping of the N-terminal alpha-helix of each monomer, prevailed over the C-dimer. Under more vigorous denaturing conditions, where also the C terminus of RNase A, richer in hydrophobic amino acids, unfolded, the C-dimer, formed by 3D domain swapping of the C-terminal beta-strand, prevailed over the other, possibly because of the induction to aggregation promoted by the hydrophobic residues present in the C termini of the two monomers.     

A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.     

Understanding how the distal environment directs reactivity in chlorite dismutase: spectroscopy and reactivity of Arg183 mutants

The chlorite dismutase from Dechloromonas aromatica (DaCld) catalyzes the highly efficient decomposition of chlorite to O(2) and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O-O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV-visible and resonance Raman spectroscopies showed that DaCld(R183A) contains five-coordinate high-spin (5cHS) heme, the DaCld(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and DaCld(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits pK(a) values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT DaCld the pK(a) of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid-base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis.     

pH effects on the stability and dimerization of procaspase-3

pH-dependent conformational changes are known to occur in dimeric procaspase-3, and they have been shown to affect the rate of automaturation. We studied the equilibrium unfolding of procaspase-3(C163S) as a function of pH (between pH 8.5 and pH 4) in order to examine these changes in the context of folding and stability. The data show that the procaspase dimer undergoes a pH-dependent dissociation below pH 5, so that the protein is mostly monomeric at pH 4. Consistent with this, the dimer unfolds via a four-state process between pH 8.5 and pH 4.75, in which the native dimer isomerizes to a dimeric intermediate, and the dimeric intermediate dissociates to a monomer, which then unfolds. In contrast, a small protein concentration dependence was observed by circular dichroism, but not by fluorescence emission, at pH 4.5 and pH 4.2. There was no protein-concentration dependence to the data collected at pH 4. Overall, the results are consistent with the redistribution of the population of native dimer (N(2)) to dimeric intermediate (I(2)) to monomeric intermediate (I), as the pH is lowered so that at pH 4, the "native" ensemble resembles the monomeric intermediate (I) observed during unfolding at higher pH. An emerging picture of the monomeric procaspase is discussed. Procaspase-3 is most stable at pH approximately 7 (24-26 kcal/mol), and while the stability decreased with pH, it was observed that dimerization contributes the majority (>70%) of the conformational free energy.     

Effect of single amino acid substitutions at positions 49 and 60 on the thermal unfolding of the tryptophan synthase alpha subunit from Salmonella typhimurium

We have used circular dichroism measurements to compare the thermal unfolding of the wild type tryptophan synthase alpha subunit from Salmonella typhimurium with that of seven mutant forms with single amino acid replacements at two active site residues. Glutamic acid 49 has been replaced by phenylalanine, glutamine, or aspartic acid. Aspartic acid 60 has been replaced by alanine, aspartic acid, asparagine, or tyrosine. Thermodynamic properties (delta G, delta H, delta S, and Tm) of the wild type and mutant forms have been determined experimentally by measuring the free energy of unfolding as a function of temperature. Increasing the pH from 7.0 to 8.8 decreases the tm of the wild type alpha subunit from 56 to 45 degrees C. The thermal unfolding of the wild type alpha subunit and of six of the seven mutant forms can be described as reversible, two-state transitions. In contrast, the melting curve of a mutant alpha subunit in which aspartic acid 60 is replaced by tyrosine indicates the presence of a folding intermediate which may correspond to a "molten globule." Correlations between our observations and previous folding studies and the X-ray crystallographic structure are presented. Substitution of glutamic acid 49, which is located in the hydrophobic "pit" of an eight-fold alpha/beta barrel, by a hydrophobic phenylalanine residue increases the tm from 56 to 60 degrees C. In contrast, replacement of aspartic acid 60, which is accessible to solvent, results in small reductions in the thermal stability.     

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.     

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.     

Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity

The small heat shock protein 27 (Hsp27 or HSPB1) is an oligomeric molecular chaperone in vitro that is associated with several neuromuscular, neurological, and neoplastic diseases. Although aspects of Hsp27 biology are increasingly well known, understanding of the structural basis for these involvements or of the functional properties of the protein remains limited. As all 11 human small heat shock proteins (sHsps) possess an α-crystallin domain, their varied functional and physiological characteristics must arise from contributions of their nonconserved sequences. To evaluate the role of two such sequences in Hsp27, we have studied three Hsp27 truncation variants to assess the functional contributions of the nonconserved N- and C-terminal sequences. The N-terminal variants Δ1-14 and Δ1-24 exhibit little chaperone activity, somewhat slower but temperature-dependent subunit exchange kinetics, and temperature-independent self-association with formation of smaller oligomers than wild-type Hsp27. The C-terminal truncation variants exhibit chaperone activity at 40 °C but none at 20 °C, limited subunit exchange, and temperature-independent self-association with an oligomer distribution at 40 °C that is very similar to that of wild-type Hsp27. We conclude that more of the N-terminal sequence than simply the WPDF domain is essential in the formation of larger, native-like oligomers after binding of substrate and/or in binding of Hsp27 to unfolding peptides. On the other hand, the intrinsically flexible C-terminal region drives subunit exchange and thermally-induced unfolding, both of which are essential to chaperone activity at low temperature and are linked to the temperature dependence of Hsp27 self-association.     

Unfolding kinetics of glutathione reductase from cyanobacterium Spirulina maxima

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

Tracking the unfolding and refolding pathways of outer membrane protein porin from Paracoccus denitrificans

We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin.     

Lipid rafts alter the stability and activity of the cholera toxin A1 subunit

Cholera toxin (CT) travels from the cell surface to the endoplasmic reticulum (ER) as an AB holotoxin. ER-specific conditions then promote the dissociation of the catalytic CTA1 subunit from the rest of the toxin. CTA1 is held in a stable conformation by its assembly in the CT holotoxin, but the dissociated CTA1 subunit is an unstable protein that spontaneously assumes a disordered state at physiological temperature. This unfolding event triggers the ER-to-cytosol translocation of CTA1 through the quality control mechanism of ER-associated degradation. The translocated pool of CTA1 must regain a folded, active structure to modify its G protein target which is located in lipid rafts at the cytoplasmic face of the plasma membrane. Here, we report that lipid rafts place disordered CTA1 in a functional conformation. The hydrophobic C-terminal domain of CTA1 is essential for binding to the plasma membrane and lipid rafts. These interactions inhibit the temperature-induced unfolding of CTA1. Moreover, lipid rafts could promote a gain of structure in the disordered, 37 °C conformation of CTA1. This gain of structure corresponded to a gain of function: whereas CTA1 by itself exhibited minimal in vitro activity at 37 °C, exposure to lipid rafts resulted in substantial toxin activity at 37 °C. In vivo, the disruption of lipid rafts with filipin substantially reduced the activity of cytosolic CTA1. Lipid rafts thus exhibit a chaperone-like function that returns disordered CTA1 to an active state and is required for the optimal in vivo activity of CTA1.     

Unfolding of rabbit muscle creatine kinase induced by acid. A study using electrospray ionization mass spectrometry,isothermal titration calorimetry,and fluorescence spectroscopy

Electrospray ionization mass spectrometry, isothermal titration calorimetry (ITC), fluorescence spectroscopy, and glutaraldehyde cross-linking SDS-PAGE have been used to study the unfolding of rabbit muscle creatine kinase (MM-CK) induced by acid. The mass spectrometric experiments show that MM-CK is unfolded gradually when titrated with acid. MM-CK is a dimer (the native state) at pH 7.0 and becomes an equilibrium mixture of the dimer and a partially folded monomer (the intermediate) between pH 6.7 and 5.0. The dimeric protein becomes an equilibrium mixture of the intermediate and an unfolded monomer (the unfolded state) between pH 5.0 and 3.0 and is almost fully unfolded at pH 3.0 reached. The results from a "phase diagram" method of fluorescence show that the conformational transition between the native state and the intermediate of MM-CK occurs in the pH range of 7.0-5.2, and the transition between the intermediate and the unfolded state of the protein occurs between pH 5.2 and 3.0. The intrinsic molar enthalpy changes for formation of the unfolded state of MM-CK induced by acid at 15.0, 25.0, 30.0, and 37.0 degrees C have been determined by ITC. A large positive molar heat capacity change of the unfolding, 8.78 kcal mol-1 K-1, at all temperatures examined indicates that hydrophobic interaction is the dominant driving force stabilizing the native structure of MM-CK. Combining the results from these four methods, we conclude that the acid-induced unfolding of MM-CK follows a "three-state" model and that the intermediate state of the protein is a partially folded monomer.     

A partially unfolded state of equine beta-lactoglobulin at pH 8.7

The urea-induced unfolding transition of equine -lactoglobulin was studied at pH 8.7 using circular dichroism (CD), ultracentrifugation, and gel filtration chromatography. The unfolding transition curves showed that at least one intermediate accumulates at moderate concentrations of urea. Furthermore, analytical ultracentrifugation experiments indicated that the intermediate forms a dimer. Thus, the urea-induced unfolding transition was measured by CD at various protein concentrations and was analyzed by a model assuming the four conformational states (the native, intermediate, dimeric intermediate, and unfolded states). The characteristics of the intermediate are markedly different from those of the intermediate previously observed at pH 4.0 or 1.5. The intermediate at pH 8.7 does not show the intense far-ultraviolet CD suggestive of the nonnative -helix.     

Linkage of proton binding to the thermal unfolding of Sso7d from the hyperthermophilic archaebacterium Sulfolobus solfataricus.

In this study the pH dependence of the thermal stability of Sso7d from Sulfolobus solfataricus is analyzed. This small globular protein of 63 residues shows a very marked dependence of thermal stability on pH: the denaturation temperature passes from 65.2 degrees C at pH 2.5 to 97.9 degrees C at pH 4.5. Analysis of the data points out that the binding of at least two protons is coupled to the thermal unfolding. By linking the proton binding to the conformational unfolding equilibrium, a thermodynamic model, which is able to describe the dependence upon the solution pH of both the excess heat capacity function and the denaturation Gibbs energy change for Sso7d, is developed. The decreased stability in very acid conditions is due to the binding of two protons on identical and noninteracting sites of the unfolded state. Actually, such sites are two carboxyl groups possessing very low pKa values in the native structure, probably involved in salt-bridges on the protein surface.