Mayhew Department of Biochemistry, University College Dublin, Bel®eld, Dublin, Ireland 1The kinetics and thermodynamics of the urea-induced unfolding of ¯avodoxin and apo¯avodoxin from D
Trang 1A comparison of the urea-induced unfolding of apo¯avodoxin
Brian OÂ Nuallain* and Stephen G Mayhew
Department of Biochemistry, University College Dublin, Bel®eld, Dublin, Ireland
1The kinetics and thermodynamics of the urea-induced
unfolding of ¯avodoxin and apo¯avodoxin from
Desulfo-vibrio 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 3 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
holo-protein is independent of urea The rates decrease in high salt
with the greater eect occurring with apoprotein The
¯uo-rescence changes ®t 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 ¯avodoxin
and apo¯avodoxin, respectively The equilibrium unfolding
curves move to greater concentrations of urea with increase
of ionic strength This eect 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 rel-ative 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 ¯uores-cence occur at low concentrations of urea and these may re¯ect binding of urea to the holoprotein
Keywords: apo¯avodoxin; urea unfolding; Desulfovibrio vulgaris
Flavodoxins are small ¯avoproteins found in
microorgan-isms 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
hydro-quinone forms of the ¯avin, and therefore the semihydro-quinone
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,
Desulfo-vibrio 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 disso-ciates ¯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 reac-tions 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 1 2837211, Tel.: + 353 1 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)
Trang 2an 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
there-fore 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
holo-protein and apoholo-protein 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]
M A T E R I A L S A N D M E T H O D S
Preparation and estimation of ¯avodoxin
and apo¯avodoxin
Flavodoxin from D vulgaris was obtained as the
recombi-nant 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
phos-phate and 0.3 mMEDTA, 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 700M)1ácm)1) and at 280 nm (22 400M)1ácm)1), respectively [13] FMN (HPLC puri®ed;
a gift from A F BuÈckmann, Gesellschaft fur Biotech-nologische Forschung Manscheroder, Weg, Germany) was determined using an absorption coef®cient of
12 500M)1ácm)1at 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 ¯uores-cence 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 1 lMprotein in urea (0.1±10M) The relatively rapid unfolding of apo¯avodoxin
in urea and 25 mMsodium phosphate buffer pH 7 (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
Fig 1 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
Trang 3signal 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
reac-tion 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
measure-ments Values for the ®rst-order rate constants for the
reactions were obtained by averaging for three
measure-ments 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
1 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
con-centration, and 20 min 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
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±5M) or by changing the phosphate buffer concentration in the range 25±250 mM, indicating that these reactions are not associated with urea-dependent unfolding Therefore, the reactions were disre-garded 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 ¯avo-doxin and apo¯avo¯avo-doxin at different concentrations of urea were also measured in refolding experiments The protein (50 lM) was unfolded in buffer B and 6Murea as described above It was then diluted 50-fold into buffer B and urea (0±6M) Refolding of the diluted protein was monitored from the changes in ¯avin and/or protein ¯uorescence 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]:
where, DGDis 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):
DGD ÿRT ln U
F
andU
F
SFÿ S
S ÿ SU 2 where S is the observed ¯uorescence signal; SFand SUare 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 SF SUeÿ DGWÿmD=RT
1 eÿ DG W ÿmD=RT 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 SFand SUfor ¯avodoxin and apo¯avodoxin are essentially independent of the urea con-centration (see below) The ®tting of an unfolding curve to
0
1
2
3
4
5
Wavelength (nm)
1
2
3
4
Fig 2 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
con-tained at 25 °C: 1 l M protein; 25 m M sodium phosphate, pH 7.0; and
for (3) and (4) 6 M urea The spectra (3) and (4) were recorded after all
¯uorescence changes were complete Fluorescence excitation was at
280 nm.
Trang 4Eqn (3) gives empirically derived m and DGWvalues without
having to determine the value for DGDat each concentration
of urea as is required when calculating a value of DGWusing
Eqn (1).In addition, the concentration ofureathatgives
half-unfolded 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 DGW nDGs;mD
where, n is the approximate number of amino-acid side
chains that become exposed on unfolding of the protein;
DGs,mis 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;
Kdenis an empirical constant that represents the
concentra-tion of denaturant at which half DGs,m is achieved The
values used for DGs,m(5.024 kJ mol)1) and Kden(25.25M)
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
com-pounds 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
D aL DL LUÿ LF 5
where Kappis the unfolding/folding equilibrium constant, aL
is the mean activity of the salt, and LUand 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 1 lM protein in buffer B and a
concentration of urea (0±1M) lower than that required to
unfold the holoprotein Equilibrium was reached within
10 min 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)
FMN-apoprotein ÿÿ*)ÿÿkoff
k on FMN apoprotein 6
Kd kkoff
on FMN ÿ apoproteinFMNapoprotein x2e
a ÿ xe 7
where Kd, is the dissociation constant; [apoprotein] and
[FMN-apoprotein], are the concentrations of free
apo¯avo-doxin 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 equilib-rium reaction (Eqn 8) [25]
kofft 2a ÿ xxe
elnx a ÿ xa xe xea
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 calcu-lated from the appropriate unfolding curves ®tted to Eqn (3) Values for konwere then calculated by substituting the values calculated for koand Kdinto Eqn (7)
Experimental data were ®tted to functions using the computer programMAC CURVEFIT(version 1.3.5)
R E S U L T S
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 transi-tion 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 accord-ing to a model for reactions that approach an equilibrium (data not shown) [25] The rate constant calculated for the reaction of apo¯avodoxin with 3 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 Further-more, when chloride ion was used to raise the ionic strength
to the same value as that of 250 mMphosphate, 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 lnkw muurea 9
Trang 5where muis 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 mMphosphate, the value determined
for kw(0.0066 s)1) is only about one third of kudetermined
in strongly denaturing conditions (ku 0.021 0.001 s)1
in 10Murea; Fig 4)
The refolding of urea-unfolded apo¯avodoxin occurs
rapidly It was complete within 2 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
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 urea-unfolding curve for 1 lM apo¯avodoxin, is 9.99 0.4
kJ mol)1 A similar experiment was carried out using
50 mMMops, 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 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 mMto 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, DGwand 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 (1.0Mguanidine HCl vs 1.35M 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
0
0.5
1
Time (s)
-4 -3 -2 -1 0
Time (s)
Fig 3 The kinetics of unfolding of apo¯avodoxin by urea The
reac-tions contained at 25 °C: 1 l M apo¯avodoxin; sodium phosphate,
pH 7.0; and urea The protein ¯uorescence was measured at 315 nm
with excitation at 280 nm d, 25 m M phosphate and 3 M urea; h,
25 m M phosphate, 500 m M NaCl and 6 M urea; m, 250 m M phosphate
and 6 M urea The inset shows the corresponding logarithmic plots.
Table 1 Eects 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 (k u ) in urea at pH 7.0 and 25 °C were determined as described in Figs 3 and 6 The errors are the standard deviations.
10 4 ´ k u (s )1 ) Flavodoxin Apo¯avodoxin
25 m M sodium phosphate 3 ± 6729 321
25 m M sodium phosphate
+ 500 m M NaCl 6 0.16 0.01 396 15
Trang 6contribution 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 6 M urea 1.42 0.02 ´
10)4s)1 (3); ku in 10M urea 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 1.81 ´ 10)4s)1) This suggests that the dissociation of
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 holo-protein suggest that the holo-protein unfolds by a simple two-state 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
Fig 5 Eects of NaCl on the urea-unfolding/folding curve for
apo-¯avodoxin Unfolding curves were determined at 25 °C for 1 l M
apo¯avodoxin in 25 m M sodium phosphate buer, pH 7, with 0 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 loga-rithm of the equilibrium constant (K app ) 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 ).
Table 2 Eects 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 1 l M protein in the buer at pH 7.0 as indicated.
Buer m(kJámol)1 á M)1) Urea( M ) 1/2 DG(kJámolw )1 )
Flavodoxin
25 m M NaP i )6.44 0.4 2.66 17.59 1.0
+ 500 m M NaCl )4.04 0.5 5.01 20.15 2.6
250 m M NaP i )6.32 0.4 4.71 29.62 2.3
Apo¯avodoxin
25 m M NaP i )7.50 0.3 1.35 9.99 0.4
+ 100 m M NaCl )6.97 0.3 2.28 15.73 0.6
+ 300 m M NaCl )7.93 0.7 2.91 22.91 2.0
-4.8
-4.4
-4
-3.6
-1 )
Urea (M)
Fig 4 The eect of urea concentration on the rate constant of unfolding
of apo¯avodoxin The logarithm of the observed ®rst-order rate
con-stant (k u ) for the unfolding of apo¯avodoxin at 25 °C in 250 m M
sodium phosphate, pH 7.0, containing 5.2±10 M urea, is plotted
against the concentration of urea The values for k u are the averages of
three kinetic traces The error bars show the standard deviations.
Trang 7(< 1Murea in buffer B) the FMN ¯uorescence increases by
up to threefold (equivalent to 3% of the FMN
¯uores-cence of fully dissociated ¯avodoxin), and comparable
changes occur in the protein ¯uorescence Several
explana-tions 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
measure-ment 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)4s)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 kois unaffected by the urea concentration, this increase
in Kdmust re¯ect a decrease in the value for the association
Table 3 Eects 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 5 and 7 using Eqn (3) Data were obtained at 25 °C in 25 m M sodium phosphate and 0±7.1 M urea.
Protein [protein]7(M) m(kJámol)1 á M)1) urea( M ) 1/2 DG(kJámolw )1 )
Flavodoxin
0.25 ) 4.79 0.4 2.44 11.68 1.1 1.00 ) 6.44 0.4 2.66 17.59 1.0 2.50 ) 7.62 0.4 2.69 21.07 1.5 5.00 ) 5.95 0.3 2.91 17.24 1.0 14.00 ) 6.22 0.4 3.28 20.62 1.4 23.00 ) 6.59 0.4 3.31 21.59 1.3 Apo¯avodoxin
0.25 ) 9.48 0.6 1.04 9.81 0.7 1.00 ) 7.50 0.3 1.35 9.99 0.4 2.50 ) 8.93 0.4 1.42 12.59 0.5 5.00 ) 7.16 0.4 1.40 10.05 0.6 23.00 ) 8.60 0.5 1.40 12.18 0.7
Fig 7 Eects of salt on the urea-unfolding curve of ¯avodoxin Unfolding curves were determined at 25 °C, pH 7, with 1 l M protein The curves were determined in 25 m M sodium phosphate without NaCl (d,s) or with 0.5 M NaCl (m), or in 250 m M 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.
Fig 6 Progress curves for the unfolding of ¯avodoxin by urea The
experiments were carried out at 25 °C, pH 7.0, with 1 l M ¯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 m M phosphate and 6 M urea; j,h,
25m M phosphate, 500 m M NaCl and 6 M urea; d, 250 m M phosphate
and 8 M urea The inset shows the corresponding logarithmic plots.
Trang 8rate 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¯avo-doxin 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 1.9M)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 0.12M
urea as judged by the extent of ¯uorescence quenching; in
contrast, the addition of holoprotein to 0.12Murea caused
less than 3% unfolding The incomplete refolding of
urea-unfolded ¯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
incu-bated in urea for up to 5 days before diluting out the
denaturant, the extent of refolding also progressively
decreases (data not shown) The urea-unfolding and
refold-ing 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 2 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
holo-protein 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 9 less than for the apoprotein
In contrast to the apoprotein for which the transition midpoint was found to be independent of the protein con-centration, the transition midpoint for the holoprotein increases gradually from 2.4Murea when the protein con-centration is 0.25 lM to 3.2M urea at 23 lM protein (Table 3) This increase may indicate that the protein asso-ciates to a polymeric form However, there is no experi-mental 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 do 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)
D I S C U S S I O N
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 ¯uores-cence emission occurs at 351 nm, and is therefore blue-shifted 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 urea-unfolding of apo¯avodoxin from Anabaena (using the data
of Fig 6 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
Table 4 Eect of urea on the rate constants and the equilibrium
disso-ciation constant for the complex of FMN and apo¯avodoxin
Experi-mental data were obtained at 25 °C with a ®nal protein concentration
of 1 l M in 25 m M sodium phosphate pH 7.0 and urea as indicated k o
and K d were determined experimentally; k on was calculated.
[urea]
( M ) 10
( M)1ás )1 ) 10
(s )1 ) 10
( M ) 0.0 8.32 1.81 2.18
0.2 5.07 2.31 4.55
0.4 4.33 1.58 3.66
0.6 3.92 1.79 4.56
0.8 3.40 2.56 7.53
1.0 2.83 2.13 7.54
Trang 9¯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
1M 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
concentra-tions of urea, but presumably also from an interaction of
urea at the FMN binding site, possibly by
hydrogen-bonding 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)1have 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
destabiliza-tion of a folding intermediate, as well as addidestabiliza-tional 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
Measure-ments of the unfolding equilibrium by additional techniques
such as far UV circular dichroism might reveal different
unfolding equilibria, as recently observed in the
guani-dine 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
stabiliza-tion 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
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 urea-unfolding 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 interac-tion 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 phos-phate 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 concen-trations 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 3 mMphosphate pH 7 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 1 min 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 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 two-state model, but rather that it occurs through an interme-diate 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
Trang 10chloride ion In support of this conclusion, the
conforma-tional stability of D vulgaris apo¯avodoxin in
guani-dine 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 (Kdvalues
of 0.1 and 0.24 nM, respectively [35,41]) It should be noted
however, that the experimental data for the D
desulfuri-cans protein do not support such a small value for the Kd
The value given in [41] was obtained from
spectrophoto-metric measurements in which aliquots of apoprotein were
added to 43.8 lMFMN It is clear that a Kdvalue as small
as 0.1 nM could not be measured by this method
Recalculation of Kdfrom the experimental points that lie
off the straight lines of Fig 4 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
mecha-nisms of unfolding of the two proteins might be
under-standable 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
desul-furicans 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
¯avo-doxin and apo¯avo¯avo-doxin 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
concentra-tions (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
(apo-urea and (apo-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
conse-quent perturbation of the holoprotein/apoprotein equilibria
(reactions A/B and G/H) Note that the equilibria A/B and
G/H do 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)
When the urea concentration is low, the small concen-tration 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-apo-FMN), 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 apo-urea complex
The scheme of Fig 8 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 pos-sible 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 equilib-rium 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
Fig 8 Scheme for the unfolding/folding reactions of ¯avodoxin in urea apo, is folded apo¯avodoxin; urea-apo-FMN is a quasi-folded ¯avo-doxin at low urea concentrations; apo-urea is quasi-folded apo¯avo-doxin 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.