No cofactor effect on equilibrium unfolding of Desulfovibrio desulfuricans flavodoxin

Flavodoxins are proteins with an alpha/beta doubly wound topology that mediate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin (K(D)=0.1 nM, oxidized FMN). To study the effect of this organic cofactor on the conformational stability, we have characterized apo and holo forms of Desulfovibrio desulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reactions for both holo- and apo-flavodoxin are reversible. However, the unfolding curves monitored by far-UV circular dichroism and fluorescence spectroscopy do not coincide. For both apo- and holo-flavodoxin, a native-like intermediate (with altered tryptophan fluorescence but secondary structure as the folded form) is present at low GuHCl concentrations. There is no effect on the flavodoxin stability imposed by the presence of the FMN cofactor (DeltaG=20(+/-2) and 19(+/-1) kJ/mol for holo- and apo-flavodoxin, respectively). A thermodynamic cycle, connecting FMN binding to folded and unfolded flavodoxin with the unfolding free energies for apo- and holo-flavodoxin, suggests that the binding strength of FMN to unfolded flavodoxin must be very high (K(D)=0.2 nM). In agreement, we discovered that the FMN remains coordinated to the polypeptide upon unfolding.     

Role of flavin mononucleotide in the thermostability and oligomerization of Escherichia coli stress-defense protein WrbA

WrbA is an oligomeric flavodoxin-like protein that binds one molecule of flavin mononucleotide (FMN) per monomer and whose redox activity is implicated in oxidative stress defense. WrbA thermostability and oligomerization in the presence and absence of bound FMN were investigated using complementary biophysical methods. Infrared spectroscopy indicates similar structures for apo and holoWrbA. FMN binding has a dramatic effect on WrbA thermal stability, shifting the Tm by approximately 40 degrees C. Upon denaturation, the protein forms insoluble aggregates that lack native secondary structure and have no bound FMN. Circular dichroism (CD) reveals that the thermal unfolding of apo and holoWrbA proceeds via the formation of an aggregation-prone intermediate that retains substantial secondary structure but has lost the native configuration of the active site. This intermediate persists in solution up to 100 degrees C at micromolar concentrations. A similar partially folded state is populated during chemical denaturation with guanidinium chloride, but accumulation of the intermediate is evident only in the absence of FMN. The results also suggest that WrbA maintains some interaction with FMN in its partially folded state, despite the loss of the induced CD signal of FMN. On the basis of these data, the unfolding process can be depicted as follows: native holoprotein --> holointermediate --> apointermediate --> insoluble aggregate. Mass spectrometry shows that FMN promotes WrbA association into tetramers, which are more thermoresistant than dimers or monomers, suggesting that multimerization underlies the FMN effect on WrbA thermostability. This study illustrates the utility of analyzing conformational transitions and intermolecular interactions using methods that probe the liquid, solid, and gas phases.     

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.     

Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.

Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein] = 1) was asymmetric about the transition temperature of 78 degrees C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 degrees C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (delta Hcal) of 200 kcal/mol. A van't Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van't Hoff enthalpies found from fits of the DSC (delta HvH = 184 kcal/mol) and spectroscopic (delta HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR in equilibrium with U+R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N in equilibrium with U). Furthermore, the NR in equilibrium with U+R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N in equilibrium with U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR in equilibrium with U+R model is the simplest model that describes the thermal unfolding of this ligand-bound protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-pressure SANS and fluorescence unfolding study of calmodulin

Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80 °C, whereas the calcium-saturated form is stable up to temperatures as high as 100 °C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000 bar (300 MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.     

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.     

Thermal unfolding of apo- and holo-enolase from Saccharomyces cerevisiae: different mechanisms, similar activation enthalpies

Yeast enolase is stabilized by its natural cofactor Mg²⁺. This stabilization is ascribed to the reduced subunit dissociation of the holoprotein. Nevertheless, how Mg²⁺ alters the unfolding mechanism has yet to be fully characterized. Here, we investigate the role of Mg²⁺ in the denaturation mechanism and unfolding kinetics of yeast enolase. Apo-enolase unfolds through a three-state process (N₂ ↔ 2I → 2D). The intermediate species is described as a monomeric molten globule-like conformation that becomes noticeable in the presence of phosphate and is able to recover its native secondary structure when cooled down. Kinetic studies confirmed the presence of the intermediate species, even though it was not noticeable in the thermal scans. The cofactor increases the cooperativity of the unfolding transitions, while the intermediate species becomes less noticeable or nonexistent. Thus, holo-enolase follows a simple two-state mechanism (N₂ → 2D). Our results indicate smaller unfolding rate-constants in the presence of Mg²⁺, thus favoring the native state. The temperature dependence of the unfolding rates allowed us to calculate the activation enthalpies of denaturation. Interestingly, despite the different unfolding mechanisms of the apo and holo forms of enolase, they both have similar activation barriers of denaturation (185-190 kJ mol⁻¹).     

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.     

Thermal unfolding of myosin rod and light meromyosin: circular dichroism and tryptophan fluorescence studies

Rabbit skeletal myosin rod, which is the coiled-coil alpha-helical portion of myosin, contains two tryptophan residues located in the light meromyosin (LMM) portion whose fluorescence contributes 27% to the fluorescence of the entire myosin molecule. The temperature dependence of several fluorescence parameters (quantum yield, spectral position, polarization) of the rod and its LMM portion was compared to the thermal unfolding of the helix measured with circular dichroism. Rod unfolds with three major helix unfolding transitions: at 43, 47, and 53 degrees C, with the 43 and 53 degrees C transitions mainly located in the LMM region and the 47 degrees C transition mainly located in the subfragment 2 region. The fluorescence study showed that the 43 degrees C transition does not involve the tryptophan-containing region and that the 47 degrees C transition produces an intermediate with different fluorescence properties from both the completely helical and fully unfolded states. That is, although the fluorescence of the 47 degrees C intermediate is markedly quenched, the tryptophyl residues do not become appreciably exposed to solvent until the 53 degrees C transition. It is suggested that although the intermediate that is formed in the 47 degrees C transition contains an extensive region which is devoid of alpha-helix, the unfolded region is not appreciably solvated or flexible. It appears to have the properties of a collapsed nonhelical state rather than a classical random coil.     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ（ｈｏｌｏ－ＳＯＤ）和脱铜锌ＳＯＤ（ａｐｏ－ＳＯＤ）在不同浓度胍溶液中的去折叠及活力变化．结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失．提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏．     

A comparative investigation of the thermal unfolding of pseudoazurin in the Cu(II)-holo and apo form

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density, and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3 degrees C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry-Eyring model: N --> U --> [corrected] F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The DeltaG value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJ.mol(-1) at 25 degrees C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emergence starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a DeltaG value of 16.5 kJ.mol(-1) at 25 degrees C. Moreover, the apo Paz unfolding occurs at 41.8 degrees C and is compatible with a two-state reversible process N --> [corrected] U.     

Guanidinium chloride induced unfolding of a hemocyanin subunit from Carcinus aestuarii. I. Apo form

The effects of guanidinium chloride (GuHCl) on the stability of the apo form of the 5S non-reassociating subunit of hemocyanin from the crab Carcinus aestuarii (apo-CaeSS2) were investigated, using a variety of optical spectroscopy techniques (light scattering (LS), fluorescence (IF and EF) and circular dichroism (CD)). The fluorescence of 8-anilino-1-naphtalene sulphonate (ANS) was strongly enhanced in the presence of apo-CaeSS2, in contrast to holo-CaeSS2, suggesting the formation of a molten globule (MG)-like state, consequent to the removal of the two copper ions from the holo subunit. Other evidences, favouring the presence of this state in apo-CaeSS2, derive from an enhanced quenching of intrinsic fluorescence (IF) by acrylamide, a higher sensibility towards aggregation and a higher IF with respect to deoxy holo-CaeSS2. Aggregation of apo-CaeSS2 below 1.2 M GuHCl was detected by LS, suggesting the formation of an aggregation-prone intermediate, called I1. Due to this effect, fluorescence and CD data could only be collected above that denaturant concentration. Both IF (protein) and EF (ANS) fluorescence data were best fitted by a two-state cooperative transition, occurring between the intermediate I1 and the unfolded state U, with C(1/2) 1.6-1.7 M. A similar two-state transition, with a slightly higher C(1/2) value (1.9 M), was also inferred from far-UV CD data, suggesting the possible formation of another intermediate. Partial refolding of apo-CaeSS2 by dilution was found to occur above 1.2 M GuHCl, i.e. up to the level of I1, since at lower denaturant concentration protein aggregation took place, as also observed in unfolding. All thermodynamic parameters, derived from data above 1.2 M GuHCl, are therefore referred to transitions between intermediate and unfolded states only. Unfolding kinetics, followed by fluorescence stopped-flow, was biphasic in the whole GuHCl range investigated (3-5 M), suggesting the formation of a transient intermediate, possibly related to that observed under equilibrium conditions.     

Cutinase unfolding and stabilization by trehalose and mannosylglycerate

The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions were coincident within experimental error when assessed by near-ultraviolet (UV) difference, tryptophyl, and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature at which 50% of cutinase is unfolded by 3 degrees C. Unfolding induced by guanidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native-like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. (Biochemistry 1993;32:3842-3851). This model takes into consideration the increase in solvation energies of the amino acid side-chains as the denaturant concentration was increased and the fraction of amino acid side-chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively.     

Isothermal unfolding studies on the apo and holo forms of Plasmodium falciparum acyl carrier protein. Role of the 4'-phosphopantetheine group in the stability of the holo form of Plasmodium falciparum acyl carrier protein

The unfolding pathways of the two forms of Plasmodium falciparum acyl carrier protein, the apo and holo forms, were determined by guanidine hydrochloride-induced denaturation. Both the apo form and the holo form displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provides values for the conformational stability of the two proteins. Although both forms have the same amino acid sequence, and they have similar secondary structures, it was found that the - DeltaG of unfolding of the holo form was lower than that of the apo form at all the temperatures at which the experiments were done. The higher stability of the holo form can be attributed to the number of favorable contacts that the 4'-phosphopantetheine group makes with the surface residues by virtue of a number of hydrogen bonds. Furthermore, there are several hydrophobic interactions with 4'-phosphopantetheine that firmly maintain the structure of the holo form. We show here for the first time that the interactions between 4'-phosphopantetheine and the polypeptide backbone of acyl carrier protein stabilize the protein. As Plasmodium acyl carrier protein has a similar secondary structure to the other acyl carrier proteins and acyl carrier protein-like domains, the detailed biophysical characterization of Plasmodium acyl carrier protein can serve as a prototype for the analysis of the conformational stability of other acyl carrier proteins.     

Association of heat-induced conformational change with activity loss of Rubisco.

Circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) were used to investigate the thermal conformational change associated with the activity loss of spinach Rubisco. CD and intrinsic fluorescence demonstrated a three stage thermal unfolding of Rubisco. At 25-45 degrees C, the secondary structure did not change but the tertiary and/or quaternary structure changed obviously with increased temperature. In 45-60 degrees C, the secondary structure showed much change with increased temperature and the tertiary and/or quaternary structure changed much faster. Over 60 degrees C, whole conformation changed abruptly with increased temperature and finally unfolded completely. DSC, CD and activity assays after annealing showed that the conformational change and the activity loss of Rubisco were completely reversible if the heating temperature was below 45 degrees C, partly reversible between 45 and 60 degrees C, and irreversible beyond 60 degrees C.     

Equilibrium and kinetics of the folding and unfolding of canine milk lysozyme

The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.     

The acid-induced state of glucose oxidase exists as a compact folded intermediate

A systematic investigation of the acid-induced unfolding of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase) (GOD) from Aspergillus niger was made using steady-state tryptophan fluorescence, circular dichroism (CD), and ANS (1-anilino 8-naphthalene sulfonic acid) binding. Intrinsic tryptophan fluorescence studies showed a maximally unfolded state at pH 2.6 and the presence of a non-native intermediate in the vicinity of pH 1.4. Flavin adenine dinucleotide (FAD) fluorescence measurements indicate that the bound cofactors are released at low pH. In the pH range studied, near- and far-UV CD spectra show maximal loss of tertiary as well as secondary structure (40%) at pH 2.6 although glucose oxidase at this pH is relatively less denatured as compared to the conformation in 6M GdnHCl. Interestingly, in the vicinity of pH 1.4, glucose oxidase shows a refolded conformation (A-state) with approximately 90% of native secondary structure and native-like near-UV CD spectral features. ANS fluorescence studies, however, show maximal binding of the dye to the protein at pH 1.4, indicating a "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a more compact conformation at low pH. Thermal stability of this state was assessed by ellipticity changes at 222 nm relative to native protein. While native glucose oxidase showed a completely reversible thermal denaturation profile, the state at pH 1.4 showed approximately 50% structural loss and the denatured state appeared to be in a different conformation exhibiting prominent beta-sheet structure (around 85 degrees C) that was not reversible. To summarize; the A-state of GOD exists as a compact folded intermediate with "molten-globule"-like characteristics, viz., native-like secondary structure but with non-native cofactor environment, enhanced hydrophobic surface area and non-cooperative thermal unfolding. That the A-state also possesses significant tertiary structure is an interesting observation made in this study.     

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.     

Thermal unfolding process of dihydrolipoamide dehydrogenase studied by fluorescence spectroscopy

The thermal unfolding pathway for dihydrolipoamide dehydrogenase (LipDH) isolated from Bacillus stearothermophilus was investigated focusing on the transient intermediate state characterized through time-resolved fluorescence studies. The decrease in ellipticity in the far UV region in the CD spectrum, the fluorescence spectral change of Trp-91 and FAD, and the thermal enzymatic inactivation curve consistently demonstrated that LipDH unfolded irreversibly on heat treatment at higher than 65 degrees C. LipDH took a transient intermediate state during the thermal unfolding process which could refold back into the native state. In this state, the internal rotation of FAD was activated in the polypeptide cage and correspondingly LipDH showed a peculiar conformation. The transient intermediate state of LipDH characterized in time-resolved fluorescence depolarization studies showed very similar properties to the molten-globule state, which has been confirmed in many studies on protein folding.