Eur J Biochem 269, 212±223 (2002) Ó FEBS 2002 A comparison of the urea-induced unfolding of apo¯avodoxin and ¯avodoxin from Desulfovibrio vulgaris  Brian O Nuallain* and Stephen G Mayhew Department of Biochemistry, University College Dublin, Bel®eld, Dublin, Ireland The kinetics and thermodynamics of the urea-induced unfolding of ¯avodoxin and apo¯avodoxin from Desulfovibrio vulgaris were investigated by measuring changes in ¯avin and protein ¯uorescence The reaction of urea with ¯avodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s)1 in M urea in 25 mM sodium phosphate at 25 °C), and it results in the dissociation of FMN The rate of unfolding of apo¯avodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea The rates decrease in high salt with the greater e€ect occurring with apoprotein The ¯uorescence changes ®t two-state models for unfolding, but they 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 ¯avodoxin and apo¯avodoxin, respectively The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength This e€ect is larger with phosphate than with chloride, and with apo¯avodoxin than with ¯avodoxin In low salt the conformational stability of the holoprotein is greater than that of apo¯avodoxin, 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 ¯avodoxin from D vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that ¯avodoxin dissociates Small changes in ¯avin ¯uorescence occur at low concentrations of urea and these may re¯ect binding of urea to the holoprotein Flavodoxins are small ¯avoproteins found in microorganisms and eukaryotic algae where they function as electron carriers in oxidation±reduction reactions [1,2] They consist of a ®ve-stranded parallel b sheet of protein with a helices on each side of the sheet, and a molecule of FMN bound strongly but noncovalently between two loops on one side of the molecule The two methyl groups on the ¯avin are exposed to solvent, the dimethylisoalloxazine moiety is ¯anked by hydrophobic residues, and the ribityl phosphate side chain extends towards the centre of the protein (Fig 1) The ¯avodoxins are believed to function as 1-electron carriers that operate between the semiquinone and hydroquinone forms of the ¯avin, and therefore the semiquinone is probably the resting state of the folded protein in the cell The ¯avodoxins occur as short-chain proteins (137±148 residues) such as those from Desulfovibrio vulgaris, Desulfovibrio desulfuricans and Clostridium beijerinckii, and a second group that has about 20 amino acids inserted in one strand of the b sheet, and that includes proteins from Azotobacter vinelandii and Anabaena PCC 7119 The ¯avodoxin fold is shared by a range of unrelated proteins (nine superfamilies) with different functions [3] The FMN of ¯avodoxins can be reversibly removed with acid The resulting apoproteins are stable, and it has been proposed that they are useful models to investigate the folding/unfolding reactions of a/b proteins [4±8] However, their ability to bind ¯avin is a property that has yet to be explored in the context of protein folding, and it is likely that they will also prove to be useful as models for folding of a/b proteins that require a tightly bound organic cofactor for activity An early study showed that guanidine HCl dissociates ¯avodoxin from Clostridium pasteurianum into apo¯avodoxin and FMN [9] More recently, this denaturant has been used to study unfolding of the apoproteins of ¯avodoxins from A vinelandii [6±8] and D desulfuricans [10], and similar studies have been carried out with apo¯avodoxin from Anabaena PCC 7119 but using urea as the denaturant [4,5] In the ®rst two cases, evidence was obtained for a stable intermediate in the equilibrium between the folded and unfolded states In contrast, urea causes apo¯avodoxin from Anabaena to unfold directly without stabilizing an intermediate The study with apo¯avodoxin from D desulfuricans was the only one to investigate the effect of bound FMN on the unfolding equilibrium It was concluded that FMN has no effect on the stability of the protein, and that FMN remains tightly bound to the unfolded protein [10] The present paper compares the unfolding/folding reactions of ¯avodoxin from D vulgaris by urea with the corresponding reactions of its apoprotein This ¯avodoxin contains two residues of tryptophan and ®ve residues of tyrosine A tyrosine side chain (Y98) is almost coplanar with the face of the dimethylisoalloxazine moiety that is closer to solvent, while a tryptophan side chain (W60) is inclined at Correspondence to S G Mayhew, Department of Biochemistry, University College Dublin, Bel®eld, Dublin 4, Ireland Fax: + 353 2837211, Tel.: + 353 7061572, E-mail: Stephen.Mayhew@UCD.IE *Present address: University of Tennessee Medical Centre, 1924 Alcoa Highway, Knoxville, TN 37920, USA (Received 10 July 2001, revised 18 October 2001, accepted 29 October 2001) Keywords: apo¯avodoxin; urea unfolding; Desulfovibrio vulgaris Ó FEBS 2002 Flavodoxin unfolding (Eur J Biochem 269) 213 Fig Structure of ¯avodoxin from D vulgaris showing the relative positions of the FMN, tryptophan side chains and tyrosine side chains The FMN is shown in yellow, tryptophan side chains in magenta, and tyrosine side chains in green The ®gure was produced with RASMOL an angle to the opposite face (Fig 1) The ¯uorescence emission by the side chains of the aromatic amino acids is partly quenched when the apoprotein binds FMN, and the intense greenish-yellow ¯uorescence of free ¯avin is about 99% quenched in the complex Measurement of the ¯uorescence emissions due to these components can therefore be used to monitor changes in the ¯avin±protein complex Changes in the ¯uorescence of the apoprotein can also provide information about the environment of the aromatic amino acids The kinetics of unfolding and refolding in urea and the unfolding equilibrium have been investigated The experimental observations have been used to determine the conformational stabilities of the holoprotein and apoprotein A mechanism is proposed for the unfolding/folding reactions of the two forms of the protein in urea This ¯avodoxin resembles ¯avodoxin from D desulfuricans both in amino-acid sequence (148 amino acids) and in three-dimensional structure [11,12] However, we ®nd that the effects of denaturant on the ¯avodoxin and apo¯avodoxin from D vulgaris are surprisingly different from those reported for the protein from D desulfuricans [10] MATERIALS AND METHODS Preparation and estimation of ¯avodoxin and apo¯avodoxin Flavodoxin from D vulgaris was obtained as the recombinant protein that was puri®ed from extracts of E coli [13] Apo¯avodoxin was prepared by acid precipitation [14] The protein precipitate was dissolved in 25 mM sodium phosphate and 0.3 mM EDTA, pH 7.0 (buffer A), and dialysed against the same buffer The concentrations of holoprotein and apoprotein were determined using absorption coef®- cients at 458 nm (10 700 M)1ácm)1) and at 280 nm (22 400 M)1ácm)1), respectively [13] FMN (HPLC puri®ed; a gift from A F Buckmann, Gesellschaft fur BiotechÈ nologische Forschung Manscheroder, Weg, Germany) was determined using an absorption coef®cient of 12 500 M)1ácm)1 at 445 nm [15] Fluorescence titrations in which apo¯avodoxin was added to a known concentration of FMN [13] showed that the concentration of apoprotein that bound FMN was at least 95% of the protein determined from the UV absorbance and that therefore the apoprotein was essentially fully active Reactions with urea The effects of urea on ¯avodoxin and its apoprotein were monitored by ¯uorimetry The protein ¯uorescence of ¯avodoxin and its apoprotein was measured with excitation at 280 nm, and emission at the wavelength that gave the greatest difference between folded and unfolded protein (380 nm and 315 nm for ¯avodoxin and apo¯avodoxin, respectively; Fig 2) The reaction of urea with ¯avodoxin was also followed by monitoring changes in ¯avin ¯uorescence at 525 nm with excitation at 445 nm (see below) Determination of unfolding rate constants Studies on the rate of protein unfolding were carried out by diluting a stock solution of ¯avodoxin or apo¯avodoxin in buffer A at least 100-fold to obtain lM protein in urea (0.1±10 M) The relatively rapid unfolding of apo¯avodoxin in urea and 25 mM sodium phosphate buffer pH (buffer B) was measured by using a stopped-¯ow spectro¯uorimeter that consisted of a Rapid Kinetics Spectrometer Accessory (Applied Photophysics Ltd; RX-1000) interfaced to the optical system of a Baird Nova ¯uorimeter, a home-made  214 B O Nuallain and S G Mayhew (Eur J Biochem 269) was followed by a further very slow change in the protein and ¯avin ¯uorescence from the holoprotein, and of the protein ¯uorescence in experiments with the apoprotein, at rates that were insigni®cant compared with the initial reaction The rates of the slow reactions were not affected by the concentration of urea (0.6±5 M) or by changing the phosphate buffer concentration in the range 25±250 mM, indicating that these reactions are not associated with ureadependent unfolding Therefore, the reactions were disregarded and the ¯uorescence at the end of the relatively rapid phase of ¯uorescence change was taken as a measure of the equilibrium between folded and unfolded protein The positions of the unfolded/folded equilibria of ¯avodoxin and apo¯avodoxin at different concentrations of urea were also measured in refolding experiments The protein (50 lM) was unfolded in buffer B and M urea as described above It was then diluted 50-fold into buffer B and urea (0±6 M) Refolding of the diluted protein was monitored from the changes in ¯avin and/or protein ¯uorescence Fluorescence (arbitrary units) 3 300 340 Ó FEBS 2002 380 420 Wavelength (nm) Fig Fluorescence emission spectra of folded and unfolded ¯avodoxin and apo¯avodoxin (1) Folded apo¯avodoxin; (2) folded ¯avodoxin; (3) unfolded ¯avodoxin; (4) unfolded apo¯avodoxin The solutions contained at 25 °C: lM protein; 25 mM sodium phosphate, pH 7.0; and for (3) and (4) M urea The spectra (3) and (4) were recorded after all ¯uorescence changes were complete Fluorescence excitation was at 280 nm signal ampli®er, an oscilloscope (Hameg Instruments 203-7) and a digital storage adaptor (Thurlby±Thandor DSA524) Progress curves for the unfolding of ¯avodoxin under all conditions, and of apo¯avodoxin at high salt concentration, were obtained by static ¯uorimetry The reaction with apo¯avodoxin was monitored continuously until the reaction was complete In contrast, the much slower unfolding reaction of ¯avodoxin was monitored at intervals; to minimize photobleaching of FMN, the reaction mixtures were stored in darkness between ¯uorescence measurements Values for the ®rst-order rate constants for the reactions were obtained by averaging for three measurements the slopes of plots of the logarithm of the change in ¯uorescence vs time Equilibrium unfolding/refolding experiments The unfolding/folding equilibrium of ¯avodoxin and apo¯avodoxin was determined by following the changes in FMN and/or protein ¯uorescence Readings were taken at intervals until equilibrium had been reached Assays were carried out in triplicate, with each mixture containing in mL at 25 °C: 0.25±23 lM protein; up to 7.1 M urea; sodium phosphate buffer pH 7.0; and NaCl as indicated The mixtures were incubated for an appropriate period (7±48 h for ¯avodoxin, depending on the phosphate concentration, and 20 for incubations with apo¯avodoxin) As is described in the Results, when the urea concentration was suf®cient to cause only partial unfolding of ¯avodoxin and apo¯avodoxin, a relatively rapid initial change occurred in the ¯uorescence until equilibrium had been reached This Analysis of the equilibrium between folded and urea-unfolded protein The urea-unfolding curves for ¯avodoxin and its apoprotein were analysed according to a two-state model that proposes that the protein unfolds in a single step The equilibrium can be ®tted to an expression of the type [16,17]: DGD ˆ DGW À mD …1† where, DGD is the change in free energy between the folded and urea-unfolded states in the denaturant; D is the concentration of denaturant; DGw is the change in free energy between the folded and unfolded states in the absence of the denaturant (the Ôconformational stabilityÕ of the protein), and, m, is an empirically derived parameter, the change in free energy between the folded and unfolded states per molar concentration of denaturant The parameter m re¯ects the cooperativity of the two-state transition Cooperativity is used to describe the sensitivity of the transition to denaturant concentration; it is not used to mean the degree to which the transition approximates a two-state transition DGD can be derived directly from experimental data (Eqn 2):   U U …SF À S† …2† and ˆ DGD ˆ ÀRT ln F F …S À SU † where S is the observed ¯uorescence signal; SF and SU are the ¯uorescence signals for the folded and unfolded protein, respectively, and F and U are the proportions of the folded and unfolded states; R is the gas constant; and T is the temperature in K The urea-unfolding curves for ¯avodoxin and apo¯avodoxin were analysed using an equation derived by Santoro & Bolen (Eqn 3) [18] that incorporates Eqns (1) and (2) S ˆ SF ‡ SU eÀ…DGW ÀmD†aRT ‡ eÀ…DGW ÀmD†aRT …3† Eqn (3) lacks parameters for the slopes of the baselines of SF and SU, which are present in the equation derived by Santoro & Bolen [18] This is because SF and SU for ¯avodoxin and apo¯avodoxin are essentially independent of the urea concentration (see below) The ®tting of an unfolding curve to Ó FEBS 2002 Flavodoxin unfolding (Eur J Biochem 269) 215 Eqn (3) gives empirically derived m and DGW values without having to determine the value for DGD at each concentration of urea as is required when calculating a value of DGW using Eqn (1) In addition, the concentration of urea that gives halfunfolded protein (urea1/2) is obtained from the ®tted curve The urea-unfolding curves were also analysed by a method [19] that is derived using experimentally measured values for the change in solvation free energy when the side chains of amino acids are transferred from water to guanidine HCl and to urea (Eqn 4) [20±23] DGD ˆ DGW ‡ nDGsYm D Kden ‡ D …4† where, n is the approximate number of amino-acid side chains that become exposed on unfolding of the protein; DGs,m is an empirically derived constant that represents an average value for the free energy change for the solvation of a buried amino-acid side chain that occurs on unfolding of the protein when the concentration of denaturant is in®nite; Kden is an empirical constant that represents the concentration of denaturant at which half DGs,m is achieved The values used for DGs,m (5.024 kJ mol)1) and Kden (25.25 M) in urea were obtained from [19] These values represent the behaviour of an ÔaverageÕ protein, determined from the solvent-excluded side chains of 55 proteins in the Protein Data Bank, and using solvation energies of model compounds in guanidine HCl and in urea [20±23] Assuming that salt ions bind preferentially to the folded state, the number of salt ions (NaCl) that are released from apo¯avodoxin when the protein unfolds can be obtained by ®tting the unfolding curves to Eqn (5) [24] D…ln Kapp † ˆ DL ˆ LU À LF D…aL † …5† where Kapp is the unfolding/folding equilibrium constant, aL is the mean activity of the salt, and LU and LF are the number of salt ions bound by the unfolded and folded states of the apoprotein, respectively Determination of kinetic and thermodynamic constants for the binding of FMN to apo¯avodoxin Rate and equilibrium constants were determined by following the increase in ¯uorescence due to FMN release when ¯avodoxin was diluted Flavodoxin was diluted at least 150-fold to obtain lM protein in buffer B and a concentration of urea (0±1 M) lower than that required to unfold the holoprotein Equilibrium was reached within 10 A value for the dissociation rate constant (ko€) was determined from the progress curve The dissociation of the holoprotein can be described by Eqns (6) and (7) koff ÀÀ FMN-apoprotein ÀÀ FMN ‡ apoprotein AB kon Kd ˆ koff ‰FMNŠ‰apoproteinŠ x2 e ˆ ˆ kon a À xe ‰FMN À apoproteinŠ …6† …7† where Kd, is the dissociation constant; [apoprotein] and [FMN-apoprotein], are the concentrations of free apo¯avodoxin and the holoprotein, respectively; a, is the initial concentration of the holoprotein and it represents 100% relative ¯uorescence if complete dissociation occurs; and xe is the concentration of FMN and apoprotein at equilibrium Solution of Eqn (7) between xi (initial), x and ti, t, yields the integrated rate law for a ®rst-order, second-order equilibrium reaction (Eqn 8) [25] koff t ˆ xe x…a À xe † ‡ xe a ln 2a À xe a…xe À x† …8† The values for xe, x, a, and t were obtained from each progress curve A plot of the right hand side of Eqn (8) vs time gives a straight line whose slope is ko€ Values for ko€ were determined from the average slope of the plots for three measurements Values for the dissociation constant for the holoprotein (Kd) were calculated from the end point of the progress curves using Eqn (8) It was assumed that the concentrations of apoprotein and free FMN in the equilibrium were the same As the experiments were carried out in a low concentration of urea, it was necessary to correct for a small proportion of unfolded apoprotein This was calculated from the appropriate unfolding curves ®tted to Eqn (3) Values for kon were then calculated by substituting the values calculated for ko€ and Kd into Eqn (7) Experimental data were ®tted to functions using the computer program MAC CURVEFIT (version 1.3.5) RESULTS Unfolding/refolding reactions of apo¯avodoxin Treatment with urea causes the ¯uorescence emission maximum for apo¯avodoxin to decrease in intensity and to shift from 336 nm to 351 nm (Fig 2) The red shift is consistent with the transfer of the aromatic residues to a more polar environment [26] and it re¯ects unfolding of the protein The ¯uorescence changes occur rapidly, and at concentrations of urea that are great enough to cause complete unfolding, the progress curve follows a single exponential (Fig 3) This suggests that a two-state transition occurs in the conversion of the folded protein to the unfolded state The reactions at concentrations of urea that are too low to cause complete unfolding were also found to follow ®rst-order kinetics when they were analysed according to a model for reactions that approach an equilibrium (data not shown) [25] The rate constant calculated for the reaction of apo¯avodoxin with M urea in buffer B is 0.67 ‹ 0.032 s)1 (Table 1) The rate depends on the ionic composition of the solution These salt effects were not examined in detail However, it was observed that when the phosphate concentration is increased from 25 mM to 250 mM, the rate constant decreases % 60-fold Furthermore, when chloride ion was used to raise the ionic strength to the same value as that of 250 mM phosphate, the decrease in the rate constant for apo¯avodoxin was somewhat less, indicating that the rate depends in addition on the nature of the salt (Table 1) The rate constant increases exponentially with increasing urea Tanford [27] proposed that the rate constant for unfolding of a protein in urea, ku, is related to the rate constant in the absence of urea, kw, and to the urea concentration by Eqn (9) ln ku ˆ lnkw ‡ mu ‰ureaŠ …9†  216 B O Nuallain and S G Mayhew (Eur J Biochem 269) ln ∆fluorescence ∆fluorescence (arbitrary units) 0.5 -1 -2 -3 -4 0 100 200 100 200 300 Time (s) 300 Time (s) Fig The kinetics of unfolding of apo¯avodoxin by urea The reactions contained at 25 °C: lM apo¯avodoxin; sodium phosphate, pH 7.0; and urea The protein ¯uorescence was measured at 315 nm with excitation at 280 nm d, 25 mM phosphate and M urea; h, 25 mM phosphate, 500 mM NaCl and M urea; m, 250 mM phosphate and M urea The inset shows the corresponding logarithmic plots where mu is proportional to the increase in exposure of the protein to solvent on going from the folded to a transition state When this equation is used to analyse the unfolding of apo¯avodoxin in 250 mM phosphate, the value determined for kw (0.0066 s)1) is only about one third of ku determined in strongly denaturing conditions (ku ˆ 0.021 ‹ 0.001 s)1 in 10 M urea; Fig 4) The refolding of urea-unfolded apo¯avodoxin occurs rapidly It was complete within s of diluting the unfolded protein 50-fold, but attempts to follow the reaction using stopped-¯ow ¯uorescence measurements were unsuccessful mainly because at this dilution, it was not possible to obtain ef®cient mixing with the instrument available The rate of change of the protein ¯uorescence is decreased when FMN is present in the diluting buffer so that the changes can be followed in a conventional ¯uorimeter Experiments in which the ®nal 20±30% of the progress curve was monitored suggest that the reaction in the presence of equimolar FMN follows second-order kinetics (16.4 ‹ 2.1 ´ 105 M)1ás)1) The rate constant was found to be similar to that observed Ó FEBS 2002 with apo¯avodoxin that had not been through the unfolding procedure (14.1 ‹ 3.1 ´ 105 M)1ás)1) Plots of the extent of ¯uorescence change at equilibrium vs the concentration of urea also suggest that in the case of this apo¯avodoxin a simple two-state transition occurs between the folded and unfolded protein (Fig 5) Dilution of the completely unfolded apoprotein with urea of different concentrations showed that the ¯uorescence at equilibrium mirrored that observed during unfolding with urea, and that the unfolding is reversible (Fig 5) The conformational stability in buffer B, determined by ®tting the ureaunfolding curve for lM apo¯avodoxin, is 9.99 ‹ 0.4 kJ mol)1 A similar experiment was carried out using 50 mM Mops, pH 7, as the buffer to allow comparison with published data for Anabaena apo¯avodoxin [4] The value obtained for the D vulgaris protein in Mops (DGw ˆ 11.71 ‹ 1.43 kJámol)1) is similar to that in phosphate, but smaller than the value for Anabaena apo¯avodoxin (17.1 ‹ 0.5 kJámol)1) Analysis of the unfolding curve for the D vulgaris apoprotein in buffer B and using Eqn (4) suggests that 39.4 ‹ 1.2 amino-acid side chains become exposed to solvent when the protein unfolds An increase in the concentration of salt causes the unfolding curve of apo¯avodoxin to shift to the right (Fig 5) The stability of the apoprotein increases about threefold when the concentration of phosphate buffer is increased from 25 mM to 250 mM (Table 2) The slope (m) in the transition region of the unfolding curve shows only small increases with increasing phosphate, the main effect being a shift of the transition midpoint Similar but slightly smaller increases in the stability of apo¯avodoxin are observed when the ionic strength is increased with NaCl (Fig 5; Table 2) By assuming that the increase in stability of apo¯avodoxin with salt is due to the preferential binding of salt to the folded protein, it can be calculated that approximately two ions are released when the apoprotein unfolds in urea (Fig 5, inset) The values of m, DGw and the concentration of urea to give half-unfolded apoprotein were found to be independent of the protein concentration (0.25±23 lM; Table 3) Equilibrium unfolding curves for apo¯avodoxin were also obtained using guanidine HCl as denaturant in buffer B The midpoint of the unfolding curve occurs at a lower concentration of denaturant (1.0 M guanidine HCl vs 1.35 M with urea) and the slope is steeper (20.7 ‹ 1.4 kJámoláM)1) The calculated conformational stability in guanidine HCl (21.15 ‹ 1.45 kJámol)1) is about twice that calculated in urea under the same conditions It seems likely that the denaturing effect of guanidine HCl is modulated by its Table E€ects of salt on the rates of urea-unfolding of ¯avodoxin and apo¯avodoxin Values for the ®rst-order rate constants for the unfolding of the protein (ku) in urea at pH 7.0 and 25 °C were determined as described in Figs and The errors are the standard deviations Bu€er 25 mM sodium phosphate 25 mM sodium phosphate + 500 mM NaCl + 250 mM NaPi [urea] (M) 104 ´ ku (s)1) Flavodoxin Apo¯avodoxin ± 1.42 ‹ 0.24 6729 ‹ 321 ± 6 0.16 ‹ 0.01 ± 0.21 ‹ 0.01 396 ‹ 15 115 ‹ 5.0 ± Ó FEBS 2002 Flavodoxin unfolding (Eur J Biochem 269) 217 -3.6 u ln k (s-1) -4 -4.4 -4.8 12 Urea (M) Fig The e€ect of urea concentration on the rate constant of unfolding of apo¯avodoxin The logarithm of the observed ®rst-order rate constant (ku) for the unfolding of apo¯avodoxin at 25 °C in 250 mM sodium phosphate, pH 7.0, containing 5.2±10 M urea, is plotted against the concentration of urea The values for ku are the averages of three kinetic traces The error bars show the standard deviations contribution to the salt concentration and a resultant increase in stabilization Unfolding/refolding reactions of ¯avodoxin Flavodoxin completely unfolds by a single exponential in a high concentration of urea, as was found to occur with apo¯avodoxin Identical kinetics are observed when the reaction is followed by measuring the protein or the ¯avin ¯uorescence (Fig 6) The ®nal ¯avin ¯uorescence is the same as that of free FMN Depending on the reaction conditions the rate constant calculated for urea-unfolding of the holoprotein is up to 5000 times smaller than that for the apoprotein (Table 1) In further contrast to the reaction with the apoprotein, the rate constant is independent of the urea concentration [ku in M urea ˆ 1.42 ‹ 0.02 ´ 10)4 s)1 (3); ku in 10 M urea ˆ 1.48 ‹ 0.05 ´ 10)4 s)1 (3)], and similar to the rate constant determined for dissociation of the holoprotein in the absence of urea (ko€ ˆ 1.81 ´ 10)4 s)1) This suggests that the dissociation of Fig E€ects of NaCl on the urea-unfolding/folding curve for apo¯avodoxin Unfolding curves were determined at 25 °C for lM apo¯avodoxin in 25 mM sodium phosphate bu€er, pH 7, with M (d), 0.1 M (r), 0.3 M (j) or 0.5 M (m) NaCl Fluorescence excitation and emission was measured at 280 nm and 315 nm, respectively The unfolding curves were ®tted using Eqn (3) A refolding curve was also determined in the absence of NaCl (half ®lled square) The data points are average values from three measurements Inset: plots of the logarithm of the equilibrium constant (Kapp) for the unfolding of apo¯avodoxin calculated at 2.94 M (j) and 3.43 M (d) urea vs the logarithm of the mean activity of NaCl (ln a‹) the holoprotein complex to apo¯avodoxin and FMN is the rate-determining step during unfolding of this ¯avodoxin Salt inhibits the rate of unfolding of ¯avodoxin but the inhibition is less than the corresponding salt inhibition observed with the apoprotein (Table 1) The equilibrium curves for urea-unfolding of the holoprotein suggest that the protein unfolds by a simple twostate transition, as was also concluded for the apoprotein The extent of the change of the ¯avin ¯uorescence at a given concentration of urea is the same as the extent of change for the protein ¯uorescence (Fig 7) It was observed that at concentrations of urea below that required to establish the folded/unfolded equilibrium Table E€ects of salt on the energetic parameters for the conformational stabilities of ¯avodoxin and apo¯avodoxin The parameters were determined at 25 °C from urea-unfolding curves for lM protein in the bu€er at pH 7.0 as indicated Bu€er Flavodoxin 25 mM NaPi + 500 mM NaCl 250 mM NaPi Apo¯avodoxin 25 mM NaPi + 100 mM NaCl + 300 mM NaCl m (kJámol)1áM)1) Urea1/2 (M) DGw (kJámol)1) )6.44 ‹ 0.4 )4.04 ‹ 0.5 )6.32 ‹ 0.4 2.66 5.01 4.71 17.59 ‹ 1.0 20.15 ‹ 2.6 29.62 ‹ 2.3 )7.50 ‹ 0.3 )6.97 ‹ 0.3 )7.93 ‹ 0.7 1.35 2.28 2.91 9.99 ‹ 0.4 15.73 ‹ 0.6 22.91 ‹ 2.0  218 B O Nuallain and S G Mayhew (Eur J Biochem 269) Ó FEBS 2002 Table E€ects of protein concentration on the energetic parameters for the conformational stabilities of ¯avodoxin and apo¯avodoxin The energetic parameters were determined by ®tting unfolding curves as in Figs and using Eqn (3) Data were obtained at 25 °C in 25 mM sodium phosphate and 0±7.1 M urea [protein] 7(M) m (kJámol)1áM)1) urea1/2 (M) DGw (kJámol)1) 0.25 1.00 2.50 5.00 14.00 23.00 ) ) ) ) ) ) 4.79 6.44 7.62 5.95 6.22 6.59 ‹ ‹ ‹ ‹ ‹ ‹ 0.4 0.4 0.4 0.3 0.4 0.4 2.44 2.66 2.69 2.91 3.28 3.31 11.68 17.59 21.07 17.24 20.62 21.59 ‹ ‹ ‹ ‹ ‹ ‹ 1.1 1.0 1.5 1.0 1.4 1.3 0.25 1.00 2.50 5.00 23.00 Protein ) ) ) ) ) 9.48 7.50 8.93 7.16 8.60 ‹ ‹ ‹ ‹ ‹ 0.6 0.3 0.4 0.4 0.5 1.04 1.35 1.42 1.40 1.40 9.81 9.99 12.59 10.05 12.18 ‹ ‹ ‹ ‹ ‹ 0.7 0.4 0.5 0.6 0.7 Flavodoxin Apo¯avodoxin (< M urea in buffer B) the FMN ¯uorescence increases by up to threefold (equivalent to % 3% of the FMN ¯uorescence of fully dissociated ¯avodoxin), and comparable changes occur in the protein ¯uorescence Several explanations for this effect were considered, including the possibility that the holoprotein partly unfolds to an intermediate in which FMN is still protein-bound, as proposed recently for the guanidine HCl unfolding of ¯avodoxin from D desulfuricans [10] However, kinetic analysis of the increase with ¯avodoxin from D vulgaris, and measurement of the ¯uorescence end point as a function of the urea concentration, suggest that the effect results mainly from a shift to the right of the holoprotein/apoprotein equilibrium described by Eqn (6) The value determined for the dissociation rate constant (ko€) is independent of the concentration of urea (2.03 ‹ 0.53 ´ 10)4 s)1 for 0±1 M urea; Table 4) The value for the holoprotein/apoprotein dissociation constant (Kd) (corrected for the proportion of apoprotein that is unfolded; see Materials and methods) increases with increasing urea in the same range (Table 4) As ko€ is unaffected by the urea concentration, this increase in Kd must re¯ect a decrease in the value for the association Fig Progress curves for the unfolding of ¯avodoxin by urea The experiments were carried out at 25 °C, pH 7.0, with lM ¯avodoxin Protein (open symbols) and ¯avin (closed symbols) ¯uorescence was measured at 380 nm and 525 nm with excitation at 280 nm and 445 nm, respectively m,n, 25 mM phosphate and M urea; j,h, 25mM phosphate, 500 mM NaCl and M urea; d, 250 mM phosphate and M urea The inset shows the corresponding logarithmic plots Fig E€ects of salt on the urea-unfolding curve of ¯avodoxin Unfolding curves were determined at 25 °C, pH 7, with lM protein The curves were determined in 25 mM sodium phosphate without NaCl (d,s) or with 0.5 M NaCl (m), or in 250 mM sodium phosphate (j); they were ®tted using Eqn (3) The plots show changes with urea concentration in the observed protein (d,m,j) and ¯avin ¯uorescence (s) Each data point is an average from three samples Ó FEBS 2002 Flavodoxin unfolding (Eur J Biochem 269) 219 Table E€ect of urea on the rate constants and the equilibrium dissociation constant for the complex of FMN and apo¯avodoxin Experimental data were obtained at 25 °C with a ®nal protein concentration of lM in 25 mM sodium phosphate pH 7.0 and urea as indicated ko€ and Kd were determined experimentally; kon was calculated [urea] (M) 10)5 ´ kon (M)1ás)1) 104 ´ ko€ (s)1) 1010 ´ Kd (M) 0.0 0.2 0.4 0.6 0.8 1.0 8.32 5.07 4.33 3.92 3.40 2.83 1.81 2.31 1.58 1.79 2.56 2.13 2.18 4.55 3.66 4.56 7.53 7.54 rate constant (kon; Table 4) It is concluded that the increase in ¯avin ¯uorescence at low urea concentrations results mainly from a direct effect of urea on the holoprotein that weakens the FMN±protein interactions and shifts the equilibrium in Eqn (6) to the right In addition, apo¯avodoxin in equilibrium Eqn (6) starts to become unfolded at these low concentrations of urea, and this contributes to dissociation of ¯avodoxin by removing folded apoprotein from the equilibrium When urea-unfolded ¯avodoxin is diluted to give a lower concentration of urea the changes in FMN and protein ¯uorescence are reversed, indicating that the unfolding is again reversible Both ¯avin and protein ¯uorescence quenching follow second order kinetics, and the rate constant for the reaction (16.4 ‹ 1.9 M)1ás)1) is identical to the value determined when unfolded apoprotein is refolded in the presence of equimolar FMN, indicating that the rate-determining step in the refolding pathway of the holoprotein is the binding of FMN However, the changes are smaller than those observed during the unfolding reaction, showing that refolding is incomplete For example, only % 80% of the holoprotein was found to refold in 0.12 M urea as judged by the extent of ¯uorescence quenching; in contrast, the addition of holoprotein to 0.12 M urea caused less than 3% unfolding The incomplete refolding of ureaunfolded ¯avodoxin is most likely due to inactivation of apoprotein during the long period required to completely unfold the holoprotein (7 h), in contrast to the much shorter period used to unfold apoprotein (20 min) This conclusion is supported by the observation that when unfolded holoprotein is exposed to urea for a longer period than is required to completely unfold the protein even less folded protein is formed when the urea is subsequently diluted out (6% recovery of folded holoprotein after 48 h unfolding) A control experiment showed that when apoprotein is incubated in urea for up to days before diluting out the denaturant, the extent of refolding also progressively decreases (data not shown) The urea-unfolding and refolding experiments were carried out in the absence of EDTA, a chelating agent that is used to protect thiol groups from heavy metal-catalysed oxidation The inclusion of mM EDTA in the incubations did not improve the reversibility of the reactions after long-term treatment with urea The conformational stability determined for the holoprotein of ¯avodoxin in buffer B (17.6 ‹ 1.0 kJámol)1, Table 3) is almost twice that of the apoprotein The greater stability of the holoprotein results from an increase in the transition midpoint which is approximately doubled The ®t of the urea-unfolding curve for ¯avodoxin with Eqn (4) suggests that the number of amino-acid side chains that become exposed when the holoprotein unfolds (30.6 ‹ 1.7) is % less than for the apoprotein In contrast to the apoprotein for which the transition midpoint was found to be independent of the protein concentration, the transition midpoint for the holoprotein increases gradually from % 2.4 M urea when the protein concentration is 0.25 lM to % 3.2 M urea at 23 lM protein (Table 3) This increase may indicate that the protein associates to a polymeric form However, there is no experimental evidence for such a phenomenon with this ¯avodoxin in the absence of urea The values calculated for the conformational stability and the slope (m) in the transition region at each protein concentration not follow a de®nite pattern (Table 2), and therefore it is not possible to draw a ®rm conclusion about the cause of this increase in the transition midpoint When the concentration of salt is increased the unfolding curve is shifted to the right (Fig 7), as also occurs with the apoprotein However, the resultant increase in stability of the holoprotein is smaller than the increase in stability that occurs with the apoprotein As a consequence, the stability of the apoprotein in high salt is greater than the stability of the holoprotein Phosphate increases the slope in the transition region of the unfolding curve for apo¯avodoxin but it has no effect on the slope of the curve for ¯avodoxin Furthermore, the slope of the curve for the holoprotein is smaller when NaCl rather than phosphate is used to increase the ionic strength (Table 2, Fig 7) DISCUSSION The experiments described in this paper show that D vulgaris apo¯avodoxin is reversibly unfolded by urea in a reaction whose equilibrium midpoint depends on the ionic composition of the solution The change in protein ¯uorescence as a function of the concentration of urea is consistent with a two-state model of unfolding, as are the kinetics of the reaction However, two observations suggest that the apoprotein of D vulgaris ¯avodoxin is not completely unfolded in urea First, the maximum ¯uorescence emission occurs at 351 nm, and is therefore blueshifted compared with the protein emission from unfolded apo¯avodoxins from A vinelandii [28] and Anabaena [4] which occurs at 354±355 nm The 3±4 nm difference suggests that the side chains of the aromatic amino acids in the urea-treated D vulgaris protein may not all be as exposed to solvent as those in the unfolded apo¯avodoxins from the other two organisms [29] Second, it is calculated that only 30% of the amino-acid side chains become exposed to solvent after urea treatment, a value that is similar to the value that can be calculated for the ureaunfolding of apo¯avodoxin from Anabaena (using the data of Fig in [4]) The observation that the ®nal protein ¯uorescence of urea-treated ¯avodoxin is the same as that of apoprotein treated with the same concentration of urea suggests that when the urea concentration is suf®ciently high both forms of the protein unfold to the same extent The observation that the ¯uorescence due to FMN in urea-unfolded  220 B O Nuallain and S G Mayhew (Eur J Biochem 269) ¯avodoxin is the same as that of protein-free FMN under the same conditions indicates that the FMN is fully dissociated from the protein The kinetics of the two unfolding reactions involve a single exponential, similar to those observed with other small single-domain proteins [29], and because the rate constants for the increase in FMN ¯uorescence and protein ¯uorescence are so similar, we conclude that the rate-limiting step in unfolding of the holoprotein is the slow dissociation of FMN from the apoprotein The only evidence so far that urea perturbs the holoprotein at urea concentrations less than that required in the transition region is the small increase in protein and ¯avin ¯uorescence at urea concentrations up to M As was discussed earlier, the ¯uorescence increase appears to be due to the release of FMN from the protein in accordance with a shift of the equilibrium in Eqn (6) to the right This results partly from unfolding of apoprotein in the holoprotein/apoprotein equilibrium at these low concentrations of urea, but presumably also from an interaction of urea at the FMN binding site, possibly by hydrogenbonding to polar groups on the protein [31] The conformational stabilities calculated for D vulgaris ¯avodoxin and apo¯avodoxin in low salt are small by comparison with other globular proteins for which values in the range 21±60 kJámol)1 have been reported [32] The value for apo¯avodoxin is also low by comparison with the values for apo¯avodoxins from Anabaena and A vinelandii The low stability of the D vulgaris protein might be because of residual structure in the urea-unfolded protein, or because of a stable intermediate not detected by ¯uorimetry that could decrease the slope in the transition region of the folding curve [17] Stabilization by salts similar to that observed with D vulgaris apo¯avodoxin and ¯avodoxin has been observed with Anabaena apo¯avodoxin [4] and with the chemotactic protein CheY that has the ¯avodoxin-like fold [33] The increase in stabilization might originate from effects such as those discussed above, including destabilization of a folding intermediate, as well as additional effects such as preferential binding of salt ions to the folded protein or an effect on the properties of the solvent The use of a single spectroscopic technique to monitor unfolding, as used in the present study, cannot exclude the formation of stable intermediates in the reaction, nor does it allow the conclusion that the protein at the end point of the transition is devoid of all secondary and tertiary structure Measurements of the unfolding equilibrium by additional techniques such as far UV circular dichroism might reveal different unfolding equilibria, as recently observed in the guanidine HCl unfolding of apo¯avodoxins from A vinelandii [6±8] and D desulfuricans [10] It is known that other proteins whose unfolding curves ®t the two-state model in fact give intermediates in their unfolding/folding reactions [34] The larger values calculated for the conformational stabilities of ¯avodoxin and its apoprotein in phosphate, compared with NaCl, and the smaller rate of unfolding of apoprotein in NaCl, indicates that the increased stabilization of the protein by salts cannot be explained simply by a shielding of charged groups that might otherwise destabilize the protein The decrease in the m value for ¯avodoxin with NaCl is unusual but not unique because a similar effect has been reported on the m value for an equilibrium intermediate in the unfolding of apomyoglobin Ó FEBS 2002 from Aplysia limacina [34] In this case it was suggested that the large decrease (% threefold) in the m value with KCl is due to deviations from a proposed three-state model It was calculated that the NaCl induced shift in the ureaunfolding curve for D vulgaris apo¯avodoxin could be due to preferential binding of two salt ions to the folded protein (Fig 5, inset) There is no direct evidence for the binding of salts by this ¯avodoxin although it is known that the rate of association of FMN with the apoprotein is inhibited by dianionic phosphate, possibly due to competition between FMN and phosphate for the binding site [35], and in the apoprotein±ribo¯avin complex a phosphate or sulfate anion occupies the site in the protein that is normally occupied by the phosphate of FMN [36] The interactions between ¯avin and apo¯avodoxin also depend on the cation; the interaction is weaker in potassium salts than in sodium salts [13,37] The multiple binding of ions to other a/b proteins and the stabilization of folded states by such ions are well established [34,38,39] According to the Hofmeister series [40], phosphate dianion and to a lesser extent chloride anion, disrupt the structure of water, markedly increase its surface tension, and decrease the solubility of nonpolar molecules (the so-called salting-out effect) If the observed salt effects on ¯avodoxin and apo¯avodoxin are due only to a change to the physical properties of the solvent, large stabilizing effects should be caused by phosphate relative to chloride Phosphate should then lead to larger shifts in the unfolding curves It is observed experimentally, however, that chloride and phosphate have similar effects, suggesting that the salt effects are not due only to a change to the physical properties of the solvent It is concluded that the greater conformational stabilities of the two forms of the protein at high concentrations of salt are probably due to a combination of factors including the preferential binding of salt ions to the folded protein, ionic strength effects, as well as to a change in the physical properties of the solvent A detailed study of unfolding of the holoprotein of a ¯avodoxin has been reported for one other protein, namely ¯avodoxin from D desulfuricans [10] This protein was unfolded with guanidine HCl in mM phosphate pH The reactions differed in several ways from the unfolding reactions of D vulgaris ¯avodoxin described above First, the reactions with the D desulfuricans holoprotein were complete in less than rather than requiring hours to reach completion Second, the protein ¯uorescence increased at low concentrations of denaturant without a change in the ¯avin ¯uorescence Third, the FMN was found to be bound tightly after the protein had been unfolded (calculated Kd ˆ 0.2 nM [10]) The changes in protein ¯uorescence were found to occur at smaller concentrations than changes in the far UV circular dichroism of the protein, leading to the conclusion that unfolding of this protein does not ®t a twostate model, but rather that it occurs through an intermediate partly unfolded state in which the FMN is still tightly bound to the apoprotein.The conformational stabilities determined for ¯avodoxin and apo¯avodoxin from D desulfuricans using guanidine HCl [10] are similar to the stabilities determined for the corresponding proteins from D vulgaris but using urea in high salt It seems likely that the charge on guanidine HCl has a stabilizing effect similar to that of a high concentration of phosphate or Ó FEBS 2002 chloride ion In support of this conclusion, the conformational stability of D vulgaris apo¯avodoxin in guanidine HCl was found to be greater than the stability in urea The marked differences between the unfolding reactions of ¯avodoxins from two species of Desulfovibrio are somewhat surprising because the primary sequences and overall crystal structures of the two proteins are very similar [11,12] Flavin binding by the apoprotein of D desulfuricans ¯avodoxin is reported to be stronger than that of the apoprotein of D vulgaris ¯avodoxin (Kd values of 0.1 and 0.24 nM, respectively [35,41]) It should be noted however, that the experimental data for the D desulfuricans protein not support such a small value for the Kd The value given in [41] was obtained from spectrophotometric measurements in which aliquots of apoprotein were added to 43.8 lM FMN It is clear that a Kd value as small as 0.1 nM could not be measured by this method Recalculation of Kd from the experimental points that lie off the straight lines of Fig in [41] indicates that its value is 0.14 ‹ 0.1 lM If the Kd value for the oxidized D desulfuricans protein is indeed % 700 times greater than that of D vulgaris ¯avodoxin, differences in the mechanisms of unfolding of the two proteins might be understandable The conformations of the two loops of protein that envelop the FMN are different in the two proteins [11,12] As a result, the carbonyl of glutamate 99 in D vulgaris ¯avodoxin points towards the solvent, while in the D desulfuricans protein it points towards the ¯avin and is 0.29 nm from O(4) of the isoalloxazine structure As was noted by others [12], the orientation in the D desulfuricans protein should lead to O-O repulsion and to a less stable ¯avin±protein complex It should be noted further that neither the published Kd value of 0.1 nM for the oxidized protein nor the re-estimated value of 0.14 lM leads to the Kd values that have been published for the semiquinone and hydroquinone forms of this ¯avodoxin [40]; the values reported seem to greatly underestimate the strengths of interaction between this apo¯avodoxin and the two reduced forms of FMN Based on the observations described above, a scheme can be devised for the unfolding/folding of D vulgaris ¯avodoxin and apo¯avodoxin in urea (Fig 8) It is proposed that urea binds rapidly to the apoprotein to form a complex to which FMN binds relatively weakly, possibly because of competition between urea and ¯avin for the same hydrogen-bonding groups on the protein and/or because of a urea-induced change in the protein conformation More denaturant binds to apoprotein at greater urea concentrations (reaction I) leading to unfolded protein [(urea)x-apo*urea] It is further proposed that when ¯avodoxin is treated with urea, the denaturant reacts rapidly with both the holoprotein (reactions E/F) and the apoprotein (reactions C/D) so that a weaker holoprotein complex results (apourea and urea-apo-FMN appear in solution) The model proposes that the subsequent unfolding/folding reactions of the holoprotein can occur by two routes One of these involves unfolding of apoprotein (reaction I) and a consequent perturbation of the holoprotein/apoprotein equilibria (reactions A/B and G/H) Note that the equilibria A/B and G/H not depend directly on the concentration of urea The other route involves further interaction of urea with the holoprotein complex (urea-apo-FMN) and the direct unfolding of this complex (reaction L) Flavodoxin unfolding (Eur J Biochem 269) 221 Fig Scheme for the unfolding/folding reactions of ¯avodoxin in urea apo, is folded apo¯avodoxin; urea-apo-FMN is a quasi-folded ¯avodoxin at low urea concentrations; apo-urea is quasi-folded apo¯avodoxin at low urea concentrations; and (urea)x-apo*-urea is unfolded protein The sum of (urea)x and urea is the concentration of urea required to completely unfold the protein When the urea concentration is low, the small concentration of apoprotein (apo-urea) that is in equilibrium with the holoprotein, unfolds rapidly to a new equilibrium that includes completely unfolded protein [(urea)x-apo*-urea], the two species of folded protein (apo-urea and urea-apoFMN), free FMN and urea The FMN prevents the apoprotein from unfolding completely and maintains a high equilibrium concentration of apo-urea-FMN As the direct unfolding/folding reactions of the holoprotein complex (reactions K/L) are very slow, the protein unfolding/folding occurs mainly via the apoprotein routes through the apourea complex The scheme of Fig provides a working hypothesis for the overall unfolding/folding reactions of D vulgaris apo¯avodoxin and ¯avodoxin, and it forms a basis for further experimentation It does not account for all of the experimental observations on the system, in particular the different effects of salt on the two forms of the protein that cause the conformational stability of the apoprotein in high salt to be greater than that of the holoprotein It is possible that an intermediate occurs during folding/unfolding of the holoprotein in high salt and that this decreases the slope of the equilibrium curve, leading to an underestimate of the conformational stability The scheme proposes that addition of free FMN should shift the unfolding equilibrium even further to the right Such a shift might be dif®cult to detect using ¯uorescence methods because of high background emission from the added ¯avin However, it should be possible to test the scheme by using an alternative method such as circular dichroism or nuclear magnetic resonance spectroscopy, together with the use of  222 B O Nuallain and S G Mayhew (Eur J Biochem 269) modi®ed ¯avins that bind to the apo¯avodoxin, and mutant apoproteins that modify the ¯avin-binding site [42] ACKNOWLEDGEMENTS We are grateful for support by Enterprise Ireland and by the EU through the Human Capital and Mobility Programme (CHRX-CT930166) We thank Dr A F Buckmann for a gift of puri®ed FMN È REFERENCES Mayhew, S.G & Ludwig, M.L (1975) Flavodoxins and ElectronTransferring Flavoproteins In The Enzymes (Boyer, P.D., ed.), Vol 12B, 3rd edn, pp 57±109 Academic Press, New York Mayhew, S.G & Tollin, G (1992) General properties of ¯avodoxins 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P.A., Yalloway, G.N & Geoghegan, S.M (1996) Regulation of the redox potentials of ¯avodoxins: modi®cation of the ¯avin binding site Biochem Soc Trans 24, 122±127  ... solvent after urea treatment, a value that is similar to the value that can be calculated for the ureaunfolding of apo¯avodoxin from Anabaena (using the data of Fig in [4]) The observation that the. .. respectively, and F and U are the proportions of the folded and unfolded states; R is the gas constant; and T is the temperature in K The urea -unfolding curves for ¯avodoxin and apo¯avodoxin were analysed... with D vulgaris apo¯avodoxin and ¯avodoxin has been observed with Anabaena apo¯avodoxin [4] and with the chemotactic protein CheY that has the ¯avodoxin- like fold [33] The increase in stabilization