Presence of the cofactor speeds up folding of Desulfovibrio desulfuricans flavodoxin

Flavodoxin is an alpha/beta protein with a noncovalently bound flavin-mononucleotide (FMN) cofactor. The apo-protein adopts a structure identical to that of the holo-form, although there is more dynamics in the FMN-binding loops. The equilibrium unfolding processes of Azotobacter vinelandii apo-flavodoxin, and Desulfovibrio desulfuricans ATCC strain 27774 apo- and holo-flavodoxins involve rather stable intermediates. In contrast, we here show that both holo- and apo-forms of flavodoxin from D. desulfuricans ATCC strain 29577 (75% sequence similarity with the strain 27774 protein) unfold in two-state equilibrium processes. Moreover, the FMN cofactor remains bound to the unfolded holo-protein. The folding and unfolding kinetics for holo-flavodoxin exhibit two-state behavior, albeit an additional slower phase is present at very low denaturant concentrations. The extrapolated folding time in water for holo-flavodoxin, approximately 280 microsec, is in excellent agreement with that predicted from the protein's native-state topology. Unlike the holo-protein behavior, the folding and unfolding reactions for apo-flavodoxin are best described by two kinetic phases, with rates differing approximately 15-fold, suggesting the presence of a kinetic intermediate. Both folding phases for apo-flavodoxin are orders of magnitude slower (40- and 530-fold, respectively) than that for the holo-protein. We conclude that polypeptide-cofactor interactions in the unfolded state of D. desulfuricans strain 29577 flavodoxin alter the kinetic-folding path towards two-state and speed up the folding reaction.     

FMN binding and unfolding of Desulfovibrio desulfuricans flavodoxin: "hidden" intermediates at low denaturant concentrations

The flavin mononucleotide (FMN) cofactor in Desulfovibrio desulfuricans flavodoxin stays associated with the polypeptide upon guanidine hydrochloride (GuHCl) induced unfolding. Using isothermal titration calorimetry (ITC), we determined the affinity of FMN for the flavodoxin polypeptide as a function of both urea and GuHCl concentrations (pH 7, 25 degrees C). The FMN affinity for folded and GuHCl-unfolded flavodoxin differs 10-fold, which is in agreement with the difference in thermodynamic stability between the apo- and holo-forms. In contrast, the urea-unfolded protein does not interact with FMN and equilibrium unfolding of holo-flavodoxin in urea results in FMN dissociation prior to polypeptide unfolding. ANS-binding, near-UV circular dichroism (CD), acrylamide quenching and FMN-emission experiments reveal the presence of native-like intermediates, not detected by far-UV CD and aromatic fluorescence detection methods, in low concentrations of both denaturants. Time-resolved experiments show that FMN binding is fastest at GuHCl concentrations where the native-like intermediate species is populated.     

Folding of Desulfovibrio desulfuricans flavodoxin is accelerated by cofactor fly-casting

Folding of cofactor-binding proteins involves ligand binding in addition to polypeptide folding. We here assess the kinetic folding/binding landscape for Desulfovibrio desulfuricans flavodoxin that coordinates an FMN cofactor. The apo-form folds in a two-step process involving a burst-phase intermediate. Studies on Tyr98Ala and Trp60Ala variants reveal that these aromatics-that stack with the FMN in the holo-form-are not participating in the apo-protein folding pathway. However, these residues are essential for FMN interactions with the unfolded protein during refolding of holo-flavodoxin. Unfolding of wild-type holo-flavodoxin is coupled to FMN dissociation whereas for Tyr98Ala and Trp60Ala holo-variants, FMN dissociates before polypeptide unfolding. Both variants refold as apo-proteins before FMN rebinds. In sharp contrast, refolding of unfolded wild-type holo-flavodoxin is over an order of magnitude faster than that of the apo-form, the pathway does not include a burst-phase intermediate, and the speed is independent of FMN excess ratio. These observations demonstrate that FMN binds rapidly to the unfolded polypeptide and guides folding straight to the native state. As this path to functional D. desulfuricans holo-flavodoxin is faster than if the cofactor binds to pre-folded apo-protein, this is one of few examples where molecular recognition via a "fly-casting" mechanism is kinetically favored.     

Native-specific stabilization of flavodoxin by the FMN cofactor: structural and thermodynamical explanation

Flavodoxins are useful models to investigate protein/cofactor interactions. The binding energy of the apoflavodoxin-FMN complex is high and therefore the holoflavodoxin is expected to be more stable than the apoprotein. This expectation has been challenged by reports on the stability of Desulfovibrio desulfuricans flavodoxin indicating that FMN binds to the unfolded polypeptide with similar affinity as to the native state, thus causing no net effect on protein stability. In previous work, we have analyzed in detail the stability of the apoflavodoxin from Anabaena PCC 7119 and the energetics of its functional complex with FMN. Here, we use the Anabaena holoprotein to directly investigate the contribution of the bound cofactor to protein stability through a detailed analysis of the chemical and thermal denaturation equilibria. Our data clearly shows that FMN binding largely stabilizes the protein towards both chemical and thermal denaturation, and that the stabilization observed at 25 degrees C in low ionic strength conditions is precisely the one expected if full release of the cofactor takes place upon flavodoxin unfolding. On the other hand, the binding of FMN to the native polypeptide is shown to simplify the thermal unfolding so that, while apoflavodoxin follows a three-state mechanism, the holoprotein unfolds in a two-state fashion. Comparison of the X-ray structure of native apoflavodoxin with the phi-structure of the thermal intermediate indicates that the increase in cooperativity driven by the cofactor originates in its preferential binding to the native state, which is a consequence of the disorganization in the intermediate of the FMN binding loops and of an adjacent longer loop.     

Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer

Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected.     

Protein folding and stability investigated by fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story

In this review, the experimental results obtained on the folding and stability of Azotobacter vinelandii flavodoxin are summarised. By doing so, three main spectroscopic techniques used to investigate protein folding and stability are briefly introduced. These techniques are: circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy in combination with the hydrogen exchange methodology. Results on the denaturant-induced and thermal equilibrium unfolding of apoflavodoxin from A. vinelandii, i.e. flavodoxin in the absence of the riboflavin-5'-monophosphate (FMN) cofactor, are discussed. A scheme for the equilibrium unfolding of apoflavodoxin is presented which involves a relatively stable molten globule-like intermediate. Denaturant-induced apoflavodoxin (un)folding as followed at the residue-level by NMR shows that the transition of native A. vinelandii apoflavodoxin to its molten globule state is highly co-operative. However, the unfolding of the molten globule to the unfolded state of the protein is non-co-operative. A comparison of the folding of A. vinelandii flavodoxin with the folding of flavodoxin from Anaboena PCC 7119 is made. The local stabilities of apo- and holoflavodoxin from A. vinelandii as measured by NMR spectroscopy are compared. Both Che Y and cutinase, which have no sequence homology with apoflavodoxin but which share the flavodoxin-like topology, have stabilisation centres different from that of apoflavodoxin from A. vinelandii. The stable centres of structurally similar proteins can thus reside in different parts of the same protein topology. Insight in the variations in (local) unfolding processes of structurally similar proteins can be used to stabilise proteins with a flavodoxin-like fold. Finally, the importance of some recent experimental and theoretical developments for the study of flavodoxin folding is briefly discussed.     

Last in, first out: the role of cofactor binding in flavodoxin folding

Although many proteins require the binding of a ligand to be functional, the role of ligand binding during folding is scarcely investigated. Here, we have reported the influence of the flavin mononucleotide (FMN) cofactor on the global stability and folding kinetics of Azotobacter vinelandii holoflavodoxin. Earlier studies have revealed that A. vinelandii apoflavodoxin kinetically folds according to the four-state mechanism: I(1) <=> unfolded apoflavodoxin <=> I(2) <=> native apoflavodoxin. I(1)an off-pathway molten globule-like is intermediate that populates during denaturant-induced equilibrium unfolding; I(2) is a high energy on-pathway folding intermediate that never populates to a significant extent. Here, we have presented extensive denaturant-induced equilibrium unfolding data of holoflavodoxin, holoflavodoxin with excess FMN, and apoflavodoxin as well as kinetic folding and unfolding data of holoflavodoxin. All folding data are excellently described by a five-state mechanism: I(1) + FMN <=> unfolded apoflavodoxin + FMN <=> I(2) + FMN <=> native apoflavodoxin + FMN<=> holoflavodoxin. The last step in flavodoxin folding is thus the binding of FMN to native apoflavodoxin. I(1),I(2), and unfolded apoflavodoxin do not interact to a significantextent with FMN. The autonomous formation of native apoflavodoxin is essential during holoflavodoxin folding. Excess FMN does not accelerate holoflavodoxin folding, and FMN does not act as a nucleation site for folding. The stability of holoflavodoxin is so high that even under strongly denaturing conditions FMN needs to be released first before global unfolding of the protein can occur.     

Thermal unfolding of Apo and Holo Desulfovibrio desulfuricans flavodoxin: cofactor stabilizes folded and intermediate states

We here compare thermal unfolding of the apo and holo forms of Desulfovibrio desulfuricans flavodoxin, which noncovalently binds a flavin mononucleotide (FMN) cofactor. In the case of the apo form, fluorescence and far-UV circular dichroism (CD) detected transitions are reversible but do not overlap (T(m) of 50 and 60 degrees C, respectively, pH 7). The thermal transitions for the holo form follow the same pattern but occur at higher temperatures (T(m) of 60 and 67 degrees C for fluorescence and CD transitions, respectively, pH 7). The holoprotein transitions are also reversible and exhibit no protein concentration dependence (above 10 microM), indicating that the FMN remains bound to the polypeptide throughout. Global analysis shows that the thermal reactions for both apo and holo forms proceed via an equilibrium intermediate that has approximately 90% nativelike secondary structure and significant enthalpic stabilization relative to the unfolded states. Incubation of unfolded holoflavodoxin at high temperatures results in FMN dissociation. Rebinding of FMN at these conditions is nominal, and therefore, cooling of holoprotein heated to 95 degrees C follows the refolding pathway of the apo form. However, FMN readily rebinds to the apoprotein at lower temperatures. We conclude that (1) a three-state thermal unfolding behavior appears to be conserved among long- and short-chain, as well as apo and holo forms of, flavodoxins and (2) flavodoxin's thermal stability (in both native and intermediate states) is augmented by the presence of the FMN cofactor.     

Flavodoxin, a new fluorescent substrate for monitoring proteolytic activity of FtsH lacking a robust unfolding activity

Escherichia coli FtsH, which belongs to the ATPases associated with diverse cellular activities (AAA) family, is an ATP-dependent and membrane-bound protease. FtsH degrades misassembled membrane proteins and a subset of cytoplasmic regulatory proteins. To elucidate the molecular mechanisms of the proteolysis, a system for precisely monitoring substrate degradation is required. We have exploited E. coli flavodoxin containing non-covalently bound flavin mononucleotide (FMN) as a model substrate for monitoring protein degradation. It was found that FtsH degrades FMN-free apo-flavodoxin but not holo-flavodoxin. However, degradation of a mutant flavodoxin carrying a substitution of Tyr94 to Asp with a lower affinity for FMN could be monitored by fluorimetry. This newly developed monitoring system will also be applicable for proteolysis by other ATP-dependent proteases.     

A comparison of the urea-induced unfolding of apoflavodoxin and flavodoxin from Desulfovibrio vulgaris.

The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s(-1) in 3 m urea in 25 mm sodium phosphate at 25 degrees C), and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding, but they do not exclude the possibility of intermediates. Calculation suggests that 21% and 30% of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin, respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride, and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin, but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein.     

Effect of inorganic phosphate on FMN binding and loop flexibility in Desulfovibrio desulfuricans apo-flavodoxin

The complex between flavin mononucleotide (FMN) and apo-flavodoxin is dominated by isoalloxazine-stacking interactions and 5'-phosphate hydrogen bonds. We show here that FMN binding to Desulfovibrio desulfuricans apo-flavodoxin is faster and the affinity is higher in the presence of inorganic phosphate as compared to in its absence (I=110 mM, pH 7, 20 degrees C). The transition-state of complex formation was investigated by phi-value analysis using Trp60Ala and Tyr98Ala apo-flavodoxin variants. We find that Tyr98 is highly involved in the FMN/protein transition state independent of inorganic phosphate, whereas the participation of Trp60 is modulated by inorganic phosphate. The phi-value for Trp60 is higher without phosphate, implying that at this condition stronger binding of Trp60 is required in the transition state to assure successful complex formation. Consistent with the experimental data, all-atom molecular dynamic simulations reveal that the presence of an anion in the phosphate subsite restricts the mobility of the Trp60-containing loop in terms of both backbone and side-chain movements, but has no effect on the Tyr98-containing loop. The overall thermodynamic stability of apo-flavodoxin is higher in the presence of inorganic phosphate as compared to in its absence (I=110 mM, pH 7, 20 degrees C). Kinetic experiments reveal that the additional stability originates in slower unfolding. The combined experimental and computational observations demonstrate that phosphate has an ordering effect on the Trp60-containing loop, which positions Trp60 favorably for FMN binding and increases the barrier for protein unfolding.     

Flavodoxin cofactor binding induces structural changes that are required for protein–protein interactions with NADP+ oxidoreductase and pyruvate formate-lyase activating enzyme

Flavodoxin (Fld) conformational changes, thermal stability, and cofactor binding were studied using circular dichroism (CD), isothermal titration calorimetry (ITC), and limited proteolysis. Thermodynamics of apo and holo-Fld folding were examined to discern the features of this important electron transfer protein and to provide data on apo-Fld. With the exception of fluorescence and UV–vis binding experiments with its cofactor flavin mononucleotide (FMN), apo-Fld is almost completely uncharacterized in Escherichia coli. Fld is more structured when the FMN cofactor is bound; the association is tight and driven by enthalpy of binding. Surface plasmon resonance binding experiments were carried out under anaerobic conditions for both apo- and holo-Fld and demonstrate the importance of structure and conformation for the interaction with binding partners. Holo-Fld is capable of associating with NADP⁺-dependent flavodoxin oxidoreductase (FNR) and pyruvate formate-lyase activating enzyme (PFL-AE) whereas there is no detectable interaction between apo-Fld and either protein. Limited proteolysis experiments were analyzed by LC-MS to identify the regions in Fld that are involved in conformation changes upon cofactor binding. Docking software was used to model the Fld/PFL-AE complex to understand the interactions between these two proteins and gain insight into electron transfer reactions from Fld to PFL-AE.     

Conformational stability of Helicobacter pylori flavodoxin: fit to function at pH 5

Flavodoxin is an essential protein for Helicobacter pylori, a pathogen living in the very acidic environment of the gastric tract and responsible for several diseases. We report the conformational stability of the protein in neutral and acidic pH. The apoprotein remains native between pH 12 and 5 and adopts a monomeric molten globule conformation at more acidic pH values. The equilibrium unfolding in urea appears two-state for either conformation, but the native one coexists with a hidden equilibrium intermediate of very similar properties. The stability of H. pylori apoflavodoxin is higher than that of the Anabaena homologue throughout the entire pH interval, which may be related to better charge compensation. H. pylori apoflavodoxin is strongly stabilized by its FMN cofactor. A global analysis of apo- and holoflavodoxin equilibrium unfolding, with and without excess FMN, indicates that the cofactor only binds to the native state. Some physical-chemical properties of the protein may represent an adaptation to the acidic environment. Unlike the apoflavodoxin from Anabaena, which becomes highly insoluble at pH 5.0, that from H. pylori remains soluble to at least 40 microm. This fact, together with the high stability of the apoprotein at this low pH that can arise in the bacteria cytoplasm, seems useful to allow newly synthesized apoflavodoxin molecules to fold and remain soluble to accomplish cofactor binding, which in turn increases the stability. Also, whenever the cytoplasmic pH drops to 5, preexisting flavodoxin molecules will remain folded and soluble and will retain the FMN cofactor, thus remaining functional.     

The flavodoxin from Helicobacter pylori: structural determinants of thermostability and FMN cofactor binding

Flavodoxin has been recently recognized as an essential protein for a number of pathogenic bacteria including Helicobacter pylori, where it has been proposed to constitute a target for antibacterial drug development. One way we are exploring to screen for novel inhibitory compounds is to perform thermal upshift assays, for which a detailed knowledge of protein thermostability and cofactor binding properties is of great help. However, very little is known on the stability and ligand binding properties of H. pylori flavodoxin, and its peculiar FMN binding site together with the variety of behaviors observed within the flavodoxin family preclude extrapolations. We have thus performed a detailed experimental and computational analysis of the thermostability and cofactor binding energetics of H. pylori flavodoxin, and we have found that the thermal unfolding equilibrium is more complex that any other previously described for flavodoxins as it involves the accumulation of two distinct equilibrium intermediates. Fortunately the entire stability and binding data can be satisfactorily fitted to a model, summarized in a simple phase diagram, where the cofactor only binds to the native state. On the other hand, we show how variability of thermal unfolding behavior within the flavodoxin family can be predicted using structure-energetics relationships implemented in the COREX algorithm. The different distribution and ranges of local stabilities of the Anabaena and H. pylori apoflavodoxins explain the essential experimental differences observed: much lower Tm1, greater resistance to global unfolding, and more pronounced cold denaturation in H. pylori. Finally, a new strategy is proposed to identify using COREX structural characteristics of equilibrium intermediate states populated during protein unfolding.     

Towards a new therapeutic target: Helicobacter pylori flavodoxin

Helicobacter pylori flavodoxin is the electronic acceptor of the pyruvate-oxidoreductase complex (POR) that catalyzes pyruvate oxidative decarboxilation. Inactivation of this metabolic route precludes bacterial survival. Because flavodoxin is not present in the human host, substances interfering electronic transport from POR might be well suited for eradication therapies against the bacterium. H. pylori flavodoxin presents a peculiar cofactor (FMN) binding site, compared to other known flavodoxins, where a conserved aromatic residue is replaced by alanine. A cavity thus appears under the cofactor that can be filled with small organic molecules. We have cloned H. pylori fldA gene, expressed the protein in Escherichia coli and characterized the purified flavodoxin. Thermal up-shift assays of flavodoxin with different concentrations of benzylamine, as well as fluorescence titration experiments indicate benzylamine binds in the pocket near the FMN binding site. It seems thus that low affinity inhibitors of H. pylori flavodoxin can be easily found that, after improvement, may give rise to leads.     

Design and Structure of an Equilibrium Protein Folding Intermediate: A Hint into Dynamical Regions of Proteins

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity.     

Ligand-linked stability of mutants of the C-domain of calmodulin

There is a necessary energetic linkage between ligand binding and stability in biological molecules. The critical glutamate in Site 4 was mutated to create two mutants of the C-domain of calmodulin yielding E140D and E140Q. These proteins were stably folded in the absence of calcium, but had dramatically impaired binding of calcium. We determined the stability of the mutant proteins in the absence and presence of calcium using urea-induced unfolding monitored by circular dichroism (CD) spectroscopy. These calcium-dependent unfolding curves were fit to models that allowed for linkage of stability to binding of a single calcium ion to the native and unfolded states. Simultaneous analysis of the unfolding profiles for each mutant yielded estimates for calcium-binding constants that were consistent with results from direct titrations monitored by fluorescence. Binding to the unfolded state was not an important energetic contributor to the ligand-linked stability of these mutants.     

Cofactor binding protects flavodoxin against oxidative stress

In organisms, various protective mechanisms against oxidative damaging of proteins exist. Here, we show that cofactor binding is among these mechanisms, because flavin mononucleotide (FMN) protects Azotobacter vinelandii flavodoxin against hydrogen peroxide-induced oxidation. We identify an oxidation sensitive cysteine residue in a functionally important loop close to the cofactor, i.e., Cys69. Oxidative stress causes dimerization of apoflavodoxin (i.e., flavodoxin without cofactor), and leads to consecutive formation of sulfinate and sulfonate states of Cys69. Use of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reveals that Cys69 modification to a sulfenic acid is a transient intermediate during oxidation. Dithiothreitol converts sulfenic acid and disulfide into thiols, whereas the sulfinate and sulfonate forms of Cys69 are irreversible with respect to this reagent. A variable fraction of Cys69 in freshly isolated flavodoxin is in the sulfenic acid state, but neither oxidation to sulfinic and sulfonic acid nor formation of intermolecular disulfides is observed under oxidising conditions. Furthermore, flavodoxin does not react appreciably with NBD-Cl. Besides its primary role as redox-active moiety, binding of flavin leads to considerably improved stability against protein unfolding and to strong protection against irreversible oxidation and other covalent thiol modifications. Thus, cofactors can protect proteins against oxidation and modification.     

Equilibrium dissociation and unfolding of the Arc repressor dimer

The equilibrium unfolding reaction of Arc repressor, a dimeric DNA binding protein encoded by bacteriophage P22, can be monitored by fluorescence or circular dichroism changes. The stability of Arc is concentration dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The stability of the protein is decreased at low pH and increased by high salt concentration. The salt dependence suggests that two ions bind preferentially to the folded protein. In 10 mM potassium phosphate (pH 7.3) and 100 mM KCl, the unfolding free energy reaches a maximum near room temperature. The results suggest that at the low protein concentrations where operator DNA binding is normally measured, Arc is predominantly monomeric and unfolded.     

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.