Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg⁸⁰-Asp⁹³ as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved.     

Evidence of stable monomeric species in the unfolding of Cu,Zn superoxide dismutase from Photobacterium leiognathi.

The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.     

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride

Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.     

Conformational changes and protein stability of the pro-apoptotic protein Bax

Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation, chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 °C as well as in solutions of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed.     

Effect of denaturants on the 11S protein of guar seed (Cyamopsis tetragonoloba)

The effect of sodium dodecyl sulphate, urea or guanidinium hydrochloride on the sedimentation velocity, viscosity, ultra violet spectra and fluorescence spectra of the 11S protein of guar seed has been determined. Sodium dodecyl sulphate dissociates the protein directly to the 2S protein, whereas urea or guanidinium hydrochloride produces an intermediate 7S protein. These reagents denature the protein also. Both the dissociative and the denaturation effect follow the order, sodium dodecyl sulphate > guanidinium hydrochloride > urea when the concentration are expressed as mols per litre. The denatured states in the three cases probably differ.     

The quaternary structure of streptavidin in urea

We report on the interactions of urea and guanidinium salts with streptavidin. Gel filtration chromatography in 0, 4, 6, and 7 M urea indicates that the streptavidin tetramer remains intact in urea. Biotin alters the electrophoretic mobility of streptavidin whether or not 6 M urea is present. The intrinsic fluorescence of streptavidin is increased and blue-shifted in 6 M urea. The fluorescence changes indicate the absence of unfolding. A conformational response to urea is possible, but much of the fluorescence change is due to urea binding as a weak biotin analog (Ka approximately 1.3 M-1). The resistance to structural perturbation by urea reflects the structural stability of streptavidin's anti-parallel beta-barrel motif. Unfolding is sluggish in 6 M guanidinium hydrochloride (half-time, approximately 50 days). After guanidinium thiocyanate unfolding, streptavidin can be refolded, but the unfolding and refolding transitions are centered at different concentrations of perturbant. Slow unfolding, with a 15th power dependence on guanidinium thiocyanate concentration, may be partially responsible for the noncoincidence of the unfolding and refolding processes. Nonequilibrium behavior is also seen in 6 M urea, as native streptavidin does not unfold and guanidinium thiocyanate unfolded streptavidin does not refold. Refolding does occur at lower concentrations of urea. Guanidinium thiocyanate only slowly unfolds the biotin-streptavidin complex. In the presence of biotin, unfolded streptavidin does not refold in 6 M guanidinium thiocyanate or in 6 M urea.     

Copper(II)-induced secondary structure changes and reduced folding stability of the prion protein

The cellular isoform of the prion protein PrP^C is a Cu²⁺-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP^C conformation are intimately linked with disease pathogenesis, the effect of Cu²⁺ ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu²⁺ ions destabilise the native fold of PrP^C. The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu²⁺. This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol^− 1 at the midpoint of unfolding). We relate Cu²⁺-induced changes in secondary structure for full-length PrP(23-231) to smaller Cu²⁺ binding fragments. In particular, Cu²⁺-induced structural changes can directly be attributed to Cu²⁺ binding to the octarepeat region of PrP^C. Furthermore, a β-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90-126) is due only to local Cu²⁺ coordination to the individual binding sites centred at His95 and His110. Cu²⁺ binding does not directly generate a β-sheet conformation within PrP^C; however, Cu²⁺ ions do destabilise the native fold of PrP^C and may make the transition to a misfolded state more favourable.     

Extensive unfolding of the C-LytA choline-binding module by submicellar concentrations of sodium dodecyl sulphate

We have investigated the stability of the choline-binding module C-LytA against sodium dodecyl sulphate (SDS)-induced unfolding at pH 7.0 and 20 degrees C. A major intermediate with an unfolded N-terminal region accumulates at around 0.75 mM SDS, whereas 2.0 mM SDS was sufficient for a complete unfolding. This might be the first report of a protein being extensively unfolded by submicellar concentrations of SDS, occurring through formation of detergent clusters on the protein surface. All transitions were reversible upon SDS complexation with beta-cyclodextrin, allowing the calculation of thermodynamic parameters. A model for the unfolding of C-LytA by SDS is presented and compared to a previous denaturation scheme by guanidine hydrochloride.     

Static and dynamic quenching of protein fluorescence. I. Bovine serum albumin.

The fluorescence parameters, lifetime, relative quantum yield, maximum and mean wavelength, half-width, and polarization, of bovine serum albumin (BSA) were measured at 15°C in aqueous solutions containing varying concentrations of different chemical perturbants, glycerol, Cu²⁺ ions, guanidine hydrochloride, and urea. By considering a quenching mechanism as being either dynamic or static, depending upon whether the quenching is or is not accompanied by a change in the fluorescence lifetime, we were able to correlate the changes produced in the various fluorescence parameters by the different chemical perturbants with changes in macromolecular structure as the concentration of perturbant was gradually increased. The addition of glycerol and of Cu²⁺ ions indicated that in aqueous BSA both tryptophan residues are below the surface of the macromolecule, out of contact with solvent water, and, as a consequence, they are statically quenched. “Ultra-Pure” guanidine hydrochloride at 2.4 M or more caused a drastic conformation change, which resulted in the emergence of a visible tyrosine peak at 304 nm in the BSA fluorescence spectrum when either 260- or 270-nm excitation was employed. With the same excitation, the enhancement of BSA tyrosine fluorescence by 6–8 M ultra-pure urea produced only a shoulder near 304 nm in the BSA fluorescence spectrum. We have introduced the use of a new relative quantum yield for protein fluorescence, q′, referenced to the quantum yield of unquenched free tryptophan, which eliminates the quenching action of water from the reference.     

Equilibrium and kinetic folding of rabbit muscle triosephosphate isomerase by hydrogen exchange mass spectrometry

Unfolding and refolding of rabbit muscle triosephosphate isomerase (TIM), a model for (betaalpha)8-barrel proteins, has been studied by amide hydrogen exchange/mass spectrometry. Unfolding was studied by destabilizing the protein in guanidine hydrochloride (GdHCl) or urea, pulse-labeling with 2H2O and analyzing the intact protein by HPLC electrospray ionization mass spectrometry. Bimodal isotope patterns were found in the mass spectra of the labeled protein, indicating two-state unfolding behavior. Refolding experiments were performed by diluting solutions of TIM unfolded in GdHCl or urea and pulse-labeling with 2H2O at different times. Mass spectra of the intact protein labeled after one to two minutes had three envelopes of isotope peaks, indicating population of an intermediate. Kinetic modeling indicates that the stability of the folding intermediate in water is only 1.5 kcal/mol. Failure to detect the intermediate in the unfolding experiments was attributed to its low stability and the high concentrations of denaturant required for unfolding experiments. The folding status of each segment of the polypeptide backbone was determined from the deuterium levels found in peptic fragments of the labeled protein. Analysis of these spectra showed that the C-terminal half folds to form the intermediate, which then forms native TIM with folding of the N-terminal half. These results show that TIM folding fits the (4+4) model for folding of (betaalpha)8-barrel proteins. Results of a double-jump experiment indicate that proline isomerization does not contribute to the rate-limiting step in the folding of TIM.     

Inactivation and Unfolding of Protein Tyrosine Phosphatase from Thermus thermophilus HB27 during Urea and Guanidine Hydrochloride Denaturation

The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.     

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

α‐Crystallin is a multimeric eye lens protein having molecular chaperone‐like function which is crucial for lens transparency. The stability and unfolding‐refolding properties of α‐crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native‐like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α‐crystallin revealed dry native‐like core of α‐crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol^?1 on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol^?1 on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α‐crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 549–560, 2014.     

Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins alphaA- and alphaB-crystallin

Oxidative stress and Cu²⁺ have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu²⁺, is also known to induce the expression of the small heat shock proteins α-crystallins. However, the role of α-crystallins in oxidative stress and in Cu²⁺-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that α-crystallins (αA- and αB-crystallin and its phosphorylation mimic, 3DαB-crystallin) bind Cu²⁺ with close to picomolar range affinity. The presence of other tested divalent cations such as Zn²⁺, Mg²⁺, and Ca²⁺ does not affect Cu²⁺ binding, indicating selectivity of the Cu²⁺-binding site(s) in α-crystallins. Cu²⁺ binding induces structural changes and increase in the hydrodynamic radii of α-crystallins. Cu²⁺ binding increases the stability of α-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of αA-crystallin increases significantly upon Cu²⁺ binding. α-Crystallins rescue amyloid beta peptide, Aβ_1-40, from Cu²⁺-induced aggregation in vitro. α-Crystallins inhibit Cu²⁺-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, α-synuclein, a Cu²⁺-binding protein, does not inhibit this oxidation process significantly. We find that the Cu²⁺-sequestering (or redox-silencing) property of α-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu²⁺ binding and redox silencing of Cu²⁺ by any heat shock protein. Thus, our study ascribes a novel functional role to α-crystallins in Cu²⁺ homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract.     

A Single Destabilizing Mutation (F9S) Promotes Concerted Unfolding of an Entire Globular Domain in γS-Crystallin

Conformational change and aggregation of native proteins are associated with many serious age-related and neurological diseases. γS-Crystallin is a highly stable, abundant structural component of vertebrate eye lens. A single F9S mutation in the N-terminal domain of mouse γS-crystallin causes the severe Opj cataract, with disruption of cellular organization and appearance of fibrillar structures in the lens. Although the mutant protein has a near-native fold at room temperature, significant increases in hydrogen/deuterium exchange rates were observed by NMR for all the well-protected β-sheet core residues throughout the entire N-terminal domain of the mutant protein, resulting in up to a 3.5-kcal/mol reduction in the free energy of the folding/unfolding equilibrium. No difference was detected for the C-terminal domain. At a higher temperature, this effect further increases to allow for a much more uniform exchange rate among the N-terminal core residues and those of the least well-structured surface loops. This suggests a concerted unfolding intermediate of the N-terminal domain, while the C-terminal domain stays intact. Increasing concentrations of guanidinium chloride produced two transitions for the Opj mutant, with an unfolding intermediate at ∼ 1 M guanidinium chloride. The consequence of this partial unfolding, whether by elevated temperature or by denaturant, is the formation of thioflavin T staining aggregates, which demonstrated fibril-like morphology by atomic force microscopy. Seeding with the already unfolded protein enhanced the formation of fibrils. The Opj mutant protein provides a model for stress-related unfolding of an essentially normally folded protein and production of aggregates with some of the characteristics of amyloid fibrils.     

Effect of denaturants on the oligomeric structure of the 11 S protein of sunflower (Helianthus annum)

The effect of sodium dodecyl sulphate, urea, guanidinium hydrochloride and heat on the oligomeric structure of the 11 S protein of sunflower has been determined. Sodium dodecyl sulphate directly dissociates the protein to 2 S subunits, whereas urea and guanidinium hydrochloride dissociate it through an intermediate 7 S protein. Heating the protein at 90‡C for 20 min caused dissociation of the 11 S protein, without any precipitation.     

Relationship between functional properties and structure of ovalbumin

The effects of ovalbumin (OVA) denaturation using urea, guanidinium chloride (GdnHCl), sodium dodecyl sulphate (SDS), cetylpyridinium chloride (CPC), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), and 5 different cationic detergents with various side chains, HCl, and CH₃COOH were observed. Progressive unfolding in ovalbumin was measured as a function of fluorescent light intensity, peak response and shift in the maximum of emission. Kinetic measurements demonstrated that the rate of denaturation usually followed a double exponential decay pattern, but at small concentrations of urea and acids first-order reaction was indicated. The reversibility of the unfolding-folding transitions was confirmed from tryptophan fluorescence and circular dichroism (CD) measurements. Differences in secondary structure were observed and changes of-helical content were calculated. Polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulphate (SDS-PAGE) showed differences in the structure of native and denatured ovalbumin. Native protein samples in PAGE demonstrated smaller number and larger mobilities of subunits than denatured ones with different reductants, such as SDS and 2-mercaptoethanol (2 ME). Scanning of SDS protein patterns showed the appearance of aggregated forms in region of 45 kD.     

Unfolding of CBP21, a lytic polysaccharide monooxygenase,without dissociation of its copper ion cofactor

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu²⁺ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, t_p, for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of t_p in the presence of excess Cu²⁺ was around 75°C, indicating that Cu²⁺ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu²⁺ ⇌ U∙Cu²⁺, where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol⁻¹ in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu²⁺ complex itself.     

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.     

Guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase: stabilization of an apo-protein by GdmCl

The guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase, was examined in detail using multiple spectroscopic techniques, enzyme activity measurements and size exclusion chromatography. The equilibrium unfolding of FprA by urea is a cooperative process where no stabilization of any partially folded intermediate of protein is observed. In comparison, the unfolding of FprA by guanidinium chloride proceeds through intermediates that are stabilized by interaction of protein with guanidinium chloride. In the presence of low concentrations of guanidinium chloride the protein undergoes compaction of the native conformation; this is due to optimization of charge in the native protein caused by electrostatic shielding by the guanidinium cation of charges on the polar groups located on the protein side chains. At a guanidinium chloride concentration of about 0.8 m, stabilization of apo-protein was observed. The stabilization of apo-FprA by guanidinium chloride is probably the result of direct binding of the Gdm+ cation to protein. The results presented here suggest that the difference between the urea- and guanidinium chloride-induced unfolding of FprA could be due to electrostatic interactions stabilizating the native conformation of this protein.     

Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu⁺ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔG_w° = 12.9 kJ mol^− 1, m = 4.1 kJ mol^− 1 M^− 1, C_m = 3 M, and ΔCp_w° = 0.93 kJ mol^− 1 K^− 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.