Alcaraz and Antonio Donaire Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez, Spain The unfolding process of the Blue Copper Protein BCP rusticyanin Rc has been
Trang 1Unfolding process of rusticyanin
Evidence of protein aggregation
Luis A Alcaraz and Antonio Donaire
Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez, Spain
The unfolding process of the Blue Copper Protein (BCP)
rusticyanin (Rc) has been studied using a wide variety of
biochemical techniques Fluorescence and CD
spectroscop-ies reveal that the copper ion plays an essential role in
sta-bilizing the protein and that the oxidized form is more
efficient than the reduced species in this respect The addition
of guanidinium chloride to Rc samples produces
aggrega-tion of the protein Gel filtraaggrega-tion chromatography and
glutaraldehyde cross-linking experiments confirm the for-mation of such aggregates Among the BCPs, this feature is exclusive to Rc The aggregation could be related to the large molecular mass and large number of hydrophobic residues
of this protein compared with those of other BCPs Keywords: aggregation; Blue Copper Protein; metalopro-tein; protein unfolding; rusticyanin
An understanding of folding processes is crucial in order
to determine the causes of protein stability [1–4] Small
proteins typically unfold by means of a simple two-state
mechanism, characterized by the absence of intermediates
between the two extreme (folded and unfolded) states The
process is usually cooperative, reflecting the complementary
nature of the tertiary interactions that maintain the protein
scaffold With larger proteins, the mechanism is more
complex, and intermediate (usually molten globule) species
appear [5–7] The existence of these states is relevant in
many biological processes such as expression of proteins,
their translocation across membranes and the possible
formation of amyloids, which, in turn, are responsible for
several neurodegenerative diseases [4,8,9] Thus, exhaustive
efforts to understand how these intermediates are formed
and their role in protein folding are being made [6,10,11]
Rusticyanin (Rc), with a molecular mass of 16.5 kDa, is
the largest Blue Copper Protein (BCP) [12,13] It is also the
most abundant protein in Acidithiobacillus ferrooxidans, a
Gram negative bacterium that extracts its energy from
oxidation of the iron(II) ion [14,15] This organism lives in
very acidic media (lower than pH 2.5) and just one of the
most remarkable features of Rc is its high stability at low pH
[16] Rc possesses an N-terminal extension (35 amino acids
in length), not present in other BCPs, that has been described
as a factor protecting the hydrophobic core of the molecule [17,18] Its role in the acid stability of the protein has also been discussed previously [19] Dynamics studies performed
by us [20] have also shown that this N-35 extension behaves like an independent module of the rest of the protein in the folded state Another intriguing property of Rc is its redox potential, 680 mV, the highest in the BCP family [21] How the protein stabilizes Cu(I) is another question that has not been resolved completely The efficiency of the protein folding in stabilizing one or both redox states is also relevant
in order to understand the mechanism of metal ion uptake and the folding mechanism itself
The unfolding process of the BCPs azurin (Az) [22–27], plastocyanin (Pc) [28–31] and pseudoazurin (PsAz) [32–34] have been characterized extensively These proteins fold according to a two-state model Thus, the kind of questions
we address in this study are the following: (a) are there any intermediate states in the Rc (un)folding process(es) and (b) which oxidation state is preferable for the folded and unfolded protein?
We present here an exhaustive study of the unfolding process of Rc Titrations of this protein (in its apo, reduced and oxidized forms) with guanidinium chloride were performed applying different techniques We demonstrate the existence of aggregates, a feature that among the BCPs is exclusive to Rc In addition, as occurs in other BCPs, the metal ion and its oxidation state are seen to be decisive in the folding and stability of Rc The results taken as a whole give
a clear picture of the unfolding process of this protein Experimental procedures
Sample preparation Recombinant rusticyanin was obtained from BL21(DE3) Escherichia colicontaining the Rc plasmid [35] Bacteria cultures were grown in suitably modified M9 medium [20] Samples (apo, reduced or oxidized Rc) for all techniques were prepared as described previously [20] Conditions for all experiments (unless otherwise indicated) were acetate
Correspondence to A Donaire, Instituto de Biologı´a Molecular y
Celular, Universidad Miguel Herna´ndez, Edificio Torregaita´n, Avda.
de la Universidad s/n, 03202-Elche (Alicante), Spain.
Fax: +34 96 6658758, Tel.: +34 96 6658942,
E-mail: adonaire@umh.es
Abbreviations: ANS, 1-anilino-8-naphthalene sulfonate; Az, azurin;
BCP, Blue Copper Protein; DOSY, Diffusion-ordered 2D NMR
spectroscopy; Pc, plastocyanin; PsAz, pseudoazurin; Rc, rusticyanin.
Note: Molecular graphic images were produced using the UCSF
CHIMERA package (http://www.cgl.ucsf.edu/chimera) from the
Com-puter Graphics Laboratory, University of California, San Francisco.
(Received 2 June 2004, revised 9 September 2004,
accepted 15 September 2004)
Trang 2buffer 10 mM, pH 5.5, 296 K Eight percent of D2O was
added to the samples used in translational diffusion
measurements In all the titrations and after the addition
of guanidinium chloride, samples were left for 15 min
before making the corresponding measurement The protein
concentration varied according to the experimental
tech-nique used: 1.2· 10)5Mfor fluorescence and CD
spectros-copies, as well as for cross-linking experiments; a 10-fold
concentration (1.2· 10)4M) for gel filtration
chromato-graphy and ANS fluorescence; and 1.5· 10)3M in
diffu-sion NMR experiments
Fluorescence spectroscopy
Fluorescence measurements were performed either on a
SLM 8000 spectrofluorimeter (Spectronics Instruments,
Urbana, IL, USA), interfaced with a Haake water bath,
or in a Cary Eclipse spectrofluorimeter (Varian, Madrid,
Spain), connected to a Pelltier cell A 1.0-cm path-length
quartz cell (Hellma QS) was used Changes in the intrinsic
fluorescence of Rc were followed by excitation at 295 nm
and its emission spectrum was recorded between 300 and
450 nm Experiments were performed with both the apo
and the Cu(II) Rc samples Cu(I) Rc titration was impeded
due to the interference produced by the reducing agent at
the wavelength of measurement
For 1-anilino-8-naphthalene sulfonate (ANS)
fluores-cence experiments in the presence of apoRc, the excitation
wavelength was set to 360 nm and the emission observed at
540 nm was recorded
Circular dichroism
CD data were collected in a Jasco J810 spectropolarimeter
with a thermostated cell holder and interfaced with a Neslab
RTE-111 water bath Spectra were obtained at a scan speed
of 20 nmÆmin)1and the average of four scans was taken
Experiments were performed with the apo or the reduced
species, using a cuvette with a path length of 0.1 cm After
each addition, the values of the ellipticity of the sample
between 260 and 200 nm were recorded For each spectrum,
the buffer baseline was subtracted
Gel filtration chromatography
Analytical gel filtration experiments with apo, Cu(I) and
Cu(II)Rc were performed Samples were incubated for
30 min in buffers containing guanidinium chloride at
concentrations of 0.0, 1.0, 2.0 or 3.0M and loaded (in
aliquots of 100 lL) into a Superdex 75 HR 10/30 column
(equilibrated previously with four column volumes of the
elution buffer) running on an AKTA FPLC system at
296 K Flow rates of 1 mLÆmin)1 were used For the
samples at a 3.0Mguanidinium chloride concentration the
flow rate was set to 0.8 mLÆmin)1to decrease the pressure
in the column This was calibrated using a gel filtration
low-molecular-mass calibration kit The standards used
and their corresponding Stokes radii were:
chymotrypsi-nogen (20.9 A˚), ovoalbumin (30.5 A˚), ribonuclease A
(16.4 A˚), and bovine serum albumin (35.5 A˚) Protein
elution was monitored by following the absorbance at
280 nm Areas of each band were integrated and then
normalized with respect to the total area of the complete experiment
The elution of a macromolecule in gel filtration experi-ments is usually given by the weight average partition coefficient (r), obtained from the expression [36]:
r¼ðVe V0Þ
Vi
ð1Þ where, Veis the elution volume of the protein, and V0and Vi are the void and the internal volumes of the column, with values of 7.48 ± 0.02 and 29.60 ± 0.06 mL, respectively The Voand Vivolumes were determined using Blue dextran (5 mgÆmL)1) andL-tryptophan (0.5 mgÆmL)1), respectively, and averaging four measurements for each agent
The partition coefficients were determined for the molecular size standards and transformed using the inverse error function complement of r (erfc)1[r]), yielding a linear relationship with the molecular Stokes radius, Rs[36,37]:
Rs¼ a þ b½erfc1ðrÞ ð2Þ where, a and b are the calibration constants for the column The relative volumes of the species corresponding to each band were estimated assuming that they are proportional to
Rs3 According to the Stokes’ law for a solvated molecule, the translational friction coefficient, f, is given by f¼ 6pgRs, where, g is the solvent viscosity The f value for an ideal unsolvated spherical molecule, f0, with the same mass and partial specific volume, is given by this expression replacing
Rs by r0 Then, the frictional coefficient of a solvated molecule, f/f0, is given by the ratio Rs/r0 For a protein, r0 can be calculated by considering that the anhydrous molecular volume (M=N) equals to the volume of a sphere:
M V
where M is the molecular mass of the protein, V is the partial specific volume of the protein and N is Avogadro’s number
If it is assumed that all deviations from unity in the frictional coefficient are due to the hydration effects an upper limit, xmax, for the hydration in grams of water bound per gram of protein is given by:
xmax¼ V
Vwater
f
f0
3
1
ð4Þ
where Vwater is the partial specific volume of water (1 cm3Æg)1)
Cross-linking experiments Intermolecular cross-linking experiments [38,39] were per-formed to assess the existence of aggregates Glutaraldehyde
is well-known to interact with the amine group of Lys residues through its two terminal carbonyl groups Thus, it links monomers by means of these amino acids converting them into oligomeric species Samples of Cu(I)Rc 1.2· 10)5Min the presence of a reducing agent (sodium dithionite) and at guanidinium chloride concentrations of 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 were incubated with
Trang 3glutaraldehyde 1% (w/v) for 1 h at room temperature and
stirred continuously Excess salt was eliminated by dialyzing
the samples against the same buffer but in the absence of a
denaturant agent Finally, proteins were resolved using
SDS/PAGE (15% w/v)
NMR experiments
Diffusion-ordered 2D NMR spectroscopy (DOSY
experi-ments) [40] were performed by using a bipolar gradient
pulse pair stimulated-echo LED sequence [41] The diffusion
labelling gradient strength was varied in the range of from
2% to 95% of 52.25 GÆcm)1 Shaped gradient pulses 2.9 ms
in duration were applied The diffusion and recycle delays
were set to 0.150 and 1.0 second, respectively A matrix data
point of 16 000· 128 was collected using 32 scans for each
experiment Water suppression was achieved by the
water-gate pulse sequence [42] The intense guanidinium chloride
signal was eliminated by presaturation The spectra were
processed applying the Laplace transform with the ILT
program included in the Bruker XWINNMR package The
intensities of the signals as a function of the gradient
strength were fitted to a bi-exponential function Relative
hydrodynamic ratios (Rh) between the folded and unfolded
species were compared using the diffusion coefficients and
the following expression [43]:
Runfh ¼dfold
dunf
where the diparameters are the diffusion coefficients, and
the scripts fold and unf refer to the folded and unfolded
species, respectively, present in the solution
The hydrodynamic radius for the folded species were
theoretically calculated by using the empirical relationship
RhD ¼ 2.21 N0.57A˚ [43], where N indicates the number of
residues For the unfolded species, the hydrodynamic radius
was estimated by using the equation RhN¼ 4.57 N0.29A˚
Once these radii were obtained, the compaction factor, C,
was calculated from the equation:
C¼R
D
h Rh
RD
h RN h
ð6Þ
where Rh is the experimental hydrodynamic radius,
obtained from the DOSY experiments, according to
Eqn (5) The limit values of the C factor, 1.0 and 0.0,
represent the completely folded and unfolded proteins,
respectively The closer the C factor to unity, the greater the
similitude of the protein to the completely folded state A
low C-value represents a conformation close to the unfolded
one [43]
Results
Fluorescence spectroscopy
Figure 1 shows the normalized change in the fluorescence
emission of apo and Cu(II)Rc at 351 nm (the unity value
was assigned arbitrarily to the emission data obtained in the
absence of guanidinium chloride) Rc possesses two
tryp-tophan residues (Trp7 and Trp127) For the apo form, a
sigmoid-like behaviour in their emission bands is found with
a middle point at a guanidinium chloride concentration of 2.1 ± 0.1M When the same titration is performed with the holoprotein in its oxidized form, no changes in the Trp environments are observed up to concentrations of 5.0Mof the denaturant agent (Fig 1) At higher concentrations, the unfolding process starts to take place The middle point of the variation is obtained for a guanidinium chloride concentration ‡ 6.5M This value is a lower limit as the data obtained at guanidinium chloride 7.2M (last experi-mental point registered) is probably not the final point of the process Only one step can be seen in this graph Thus, either only one of the Trp residues changes its local environment
or both residues experiment a similar modification simul-taneously The parallelism between these data and those obtained by CD (see below) makes this last hypothesis more plausible Comparison between apo and holoforms (Fig 1) shows the stabilizing effect of the metal ion
Rc in the presence of ANS (data not shown) does not produce any appreciable variation in the fluorescence probe
It is well established that ANS binds to molten globule species where hydrophobic cores are exposed to the solvent and, thus, to the probe [44] Then, if such molten globule species exist, they are not accessible to the solvent
CD titrations
CD titrations were carried out with apo and Cu(I)Rc Figure 2A displays some CD spectra of the titration for apoRc As can be seen, at a high guanidinium chloride concentration (4.0M), the spectrum obtained reflects the typical random coil conformation (without any residual secondary structure elements), indicating that the complete unfolding process has already been completed Figure 2B displays the fraction of the unfolded protein according to the change observed in the ellipticity at 215 nm for the apoprotein Fifty percent of the protein was unfolded at a guanidinium chloride 2.4M, close to the value found by fluorescence spectroscopy (similar results were obtained with the ellipticity values at 222 nm, data not shown) This probably reflects that the same phenomena are observed with the two techniques
In the same Figure, the titration of the reduced protein is also shown A midpoint of 6.3 ± 0.2 guanidinium
Fig 1 Relative fluorescence emission of Rc at 351 nm vs guanidinium chloride concentration Data correspond to the apo (d), and the oxidized (s) protein.
Trang 4chloride is found by following the ellipticity at 215 nm.
Thus, CD (as does fluorescence) spectroscopy also reveals
that copper ion stabilizes the folded vs the unfolded state as
compared with the apoform
Attempts to perform a complete titration with the
oxidized protein were not successful First, no variations
were found up to guanidinium chloride concentrations of
6.0M For larger denaturant concentrations,
experi-ments were not reproducible, probably due to irreversible
reactions between the copper(II) ion and a thiol atom of
the cysteine ligand, as reported previously for azurin [23]
However, as Cu(I)Rc shows measurable changes in the
CD spectra for concentrations lower than 6.0M in
guanidinium chloride (a decrease of 33%, Fig 2B), it
may be deduced clearly that the oxidized form of Rc
possesses higher stability than the reduced species with
regard to the unfolding process
Gel filtration experiments
In order to acquire information about the shape, the volume
and the molecular mass of the folded and unfolded species,
gel filtration chromatography experiments were performed
Figure 3 shows the results obtained for Cu(I)Rc Two
features stand out from these data First, the main elution
volume (bands marked with upper case letters in Fig 3)
decreases when guanidinium chloride is increased The small
differences in the elution volumes among these peaks
suggest that these bands are due to monomeric species
(Stokes radius and C compact factors calculated from them
confirm this suggestion, see below) These bands can arise either from an increment in the hydrodynamic volume of the folded species (as a consequence of the presence of the denaturant agent), or from an averaging effect between the folded and the denaturant species Second, two bands, corresponding to much shorter elution volumes (marked with lower cases without and with commas in Fig 3) appear weakly at guanidinium chloride 1.0M and are clearly observed at 3.0M The elution volumes of these bands indicate that they proceed from species with volumes much larger than that of the monomeric form, i.e they belong to aggregates
Analogous results were obtained for the apo and the Cu(II)Rc species These results are summarized in Table 1
As can be seen, slightly higher concentrations of guanidi-nium chloride are required for the same effects to occur in the oxidized protein as in the reduced species For apoRc, lower quantities of denaturant agent are needed Moreover,
at the same concentration of guanidinium chloride, the normalized areas of the bands corresponding to aggregates (c and c¢ lower case letters) are greater for apoRc than for the two holoforms of Rc In other words, apoRc stability against unfolding is lower than that of the holoprotein Within the metallated species, Cu(I)Rc possesses lower stability than Cu(II)Rc
Fig 2 Circular dichroism titrations (A) CD spectra of apoRc
titra-tion with guanidinium chloride (B) Relative ellipticity of Rc at 215 nm
vs the guanidinium chloride concentration Data correspond to the
apo (d), and the reduced (s) protein.
Fig 3 Gel filtration elution bands for Cu(I)Rc Guanidinium chloride concentrations were 0.0 (A band), 1.0 (B and b), 2.0 (C, c and c¢) and 3.0 M (D, d and d ¢) Uppercase letters refer to the monomer (essentially folded) species Lowercase letters without and with commas refer to bands that correspond to aggregated species Upper panel: complete elution filtration Lower panel: expansion corresponding to bands of aggregated species *Bands of the void volume of the column.
Trang 5The Stokes radius of the species producing each band
(Table 1) was calculated using Eqn (2) Assuming a
similar shape, it is possible to estimate the relative
volumes of bands b, c and d These peaks correspond
to aggregates of volumes between three and four times the
volume of their corresponding folding species at the same
guanidinium chloride concentration (B, C and D,
respect-ively, Table 1) These volumes are roughly concordant
with the existence of tetramers On the same basis,
volumes of approximately eight monomers per molecule
are estimated for bands c¢ and d ¢ (Table 1) The precise
results and the concordance among them for the three
forms of Rc confirm the correctness of the assumed
hypothesis put forward (similar volume) and of the
conclusion drawn (existence of tetra and octamers)
It is noteworthy that the degree of hydration is low in the
folded species, with xmaxvalues of 0.21, 0.21 and 0.34 for
the apo, Cu(I), and Cu(II) (A bands in Table 1),
respect-ively This indicates that the Cu(I) form is found in a more
apolar environment, i.e Cu(II) is stabilized in some way by
this excess of water molecules It has been suggested that this
is a relevant factor in the high redox potential of Rc [45]
The present data are in agreement with this hypothesis However, when the protein starts to open, the degree
of hydration is nearly the same in the three species (xmax 1.1–1.2 for C and 1.3–1.4 for D bands, Table 1) Thus, in this more opened state, exposure to the solvent is similar in the three species independent of the existence and oxidation state of the metal ion
Intermolecular cross-linking Figure 4 shows the result of glutaraldehyde cross-linking experiments [38,39] on a Cu(II)Rc sample at different denaturant concentrations The pattern observed is very similar for guanidinium chloride concentrations between 1.0 and 4.0M Three bands that correspond to a monomer,
a dimer (the most intense band) and a tetramer (the weakest band) appear For higher guanidinium chloride concentrations, the band corresponding to the dimer is attenuated and those of the monomer and tetramer increase their intensity Therefore, this technique also reveals the existence of aggregates, as observed by gel filtration experiments
Table 1 Peaks observed in gel filtration experiments The experiments were performed at different concentrations of guanidinium chloride for the apo, the Cu(I) and the Cu(II) Rc The denomination of the elution bands is the same as in the caption to Fig 3 R S values were obtained by applying Eqn (2) Upper limits for hydration, x max , have only been calculated for monomer species C compact factors were calculated from the R S values and from the DOSY experiments according to Eqn (6).
Band
[Guanidinium
chloride] (M) V e (mL) R S (A˚) x max V rel A rel (%) C factor ApoRc
Cu(I)Rc
Cu(II) Rc
a
Relative volumes of b, c and d bands, on one hand, and c¢ and d ¢ bands, on the other, have been calculated with regard to volumes of peaks
B, C and D, respectively.
Trang 6Translational diffusion measurements
When DOSY measurements were performed in an apoRc
sample containing guanidinium chloride at 2.1M(data not
shown), only two species (in slow exchange regime) were
distinguished They both belong to monomer species as the
observation of aggregates is precluded by fast transversal
relaxation The ratio between the diffusion coefficients for
these two species was 1.9 Assuming an Rfold
h value of 18.3 A˚
for the Cu(I)Rc folded protein (Table 1), an Runf
h value of 34.8 A˚ is obtained for the unfolded species (Eqn 5) This
high increment in the volume upon denaturation is probably
related to a high increment in the solvation effect, and to a
change in the shape of the totally unfolded species
Using the empirical relationships described in the
Experi-mental procedures [43], radii of 20.5 and 39.2 A˚ for the
folded and unfolded species, respectively, are calculated
The first value agrees roughly with that obtained from the
DOSY experiments The second one indicates that the
spe-cies present are not 100% unfolded In fact, the compaction
factor (Eqn 6) calculated from these values was 0.24 This
result would indicate that the species observed are almost,
but not completely, unfolded
Discussion
Existence of aggregates
The present results obtained using different techniques
confirm the existence of protein aggregation in the presence
of guanidinium chloride Gel filtration experiments show
that the aggregated species contain four or eight monomers
per molecule (Table 1) Cross-linking experiments also
reveal the existence of aggregates, although, according to
this technique, they would have half the number of
molecules (Fig 4) This apparent contradiction could arise
due to two different reasons First, the glutaraldehyde
cross-linking technique is only qualitative and its results are not as
precise as those obtained with gel filtration experiments The
glutaraldehyde reaction is irreversible, and so the nature and
quantity of the products strongly depend on the incubation
time (among other factors) However, another explanation
can be found if we inspect the three dimensional structure of
Rc, shown in Fig 5 In fact, all the Lys residues of Rc are found on one (the most polar) face, while the other face is essentially nonpolar and rich in hydrophobic residues It is probable that glutaraldehyde links the dimers whose polar sides are facing Apolar (non-cross-linked) sides could be disrupted when SDS is added to the solution (prior to electrophoresis) If so, polymers observed by this technique would possess half the number of monomers they actually have in solution We believe that the results obtained here from cross-linking experiments are probably due to a combination of both these factors
Hydrophobic patches in BCPs [46–50] are related to the recognition of the proteins by their redox partners This situation could be similar for Rc [51] It is also noteworthy that a mutant of Rc with the first 35 amino acids deleted (N-35 Rc) also forms aggregates in solution [19] One should bear in mind that this N-35 deletion leaves the hydrophobic residues more exposed to the solvent Mobility data for the folded Rc have shown that this domain behaves independ-ently to the rest [20] So, it is likely that Rc with an open structure existing in the presence of guanidinium chloride could facilitate hydrophobic interactions The negative response of ANS fluorescence indicates that the hydropho-bic residues are not exposed to the solvent, i.e they have to interact with analogous residues of other protein molecules Then, aggregation is induced
Fluorescence (Fig 1) and CD (Fig 2) spectroscopies reveal that the secondary structure and tryptophan sur-roundings are essentially unaltered up to high guanidinium chloride concentrations for the holoprotein ( 5.0M) Thus, if aggregates are being formed at lower concentra-tions, they basically possess the same secondary and tertiary structure as the folded state Only when guanidinium chloride is high enough do unfolded species appear and then, the three dimensional structure of Rc is modified The existence of aggregates as intermediates in the process of unfolding has been proposed for several proteins [2,4,6,52] With our present data, we cannot state if these aggregates are intermediates in the unfolding process or an
off-pathway product of the unfolding process Previously, studies on other BCPs, specifically Pc [29–31], Az [22,23,27]
Fig 5 Hydrophobic and hydrophilic faces of rusticyanin Red colors indicate hydrophobic residues Blue colors indicate the position of Lys residues (right side) Coordinates of Rc were obtained from the protein database (pdb) file 1cur [18] The green arrow indicates the position of the copper ion (hidden from the solvent, inside the hydrophobic core, left side) The drawing was created with the CHIMERA program [62].
Fig 4 SDS/PAGE electrophoresis of Cu(II)Rc incubated previously in
presence of glutaraldehyde at different concentrations of guanidinium
chloride Solid and dotted arrows indicate the position of the dimer and
tetramer species, respectively (see text).
Trang 7and PsAz [33], have shown that in these the unfolding
process obeys a two-step model No intermediate aggregates
have been described in their unfolding processes What are
the structural features that cause the different pattern of Rc?
First, we have to keep in mind that Rc is the largest BCP
Then, the N-35 terminal extension (not present in the rest of
the BCPs) may play an important role The existence of
clearly differentiated domains could be one of the factors in
the singular behavior of Rc Second, Rc possesses a high
content of hydrophobic residues Moreover, from D2O/
H2O exchange experiments in their folded states, it has been
shown that the content of residues hidden from the solvent
is much higher in Rc than in other BCPs [20] It makes sense
that the interaction among these hydrophobic residues
facilitates the formation of aggregates when the protein
starts to open (as a consequence of the presence of the
denaturant agent)
Metal ion, oxidation state and stability
Fluorescence and CD spectroscopies reveal unequivocally
that holoforms are more efficient at stabilizing the folded
protein than the apoform Of these, the oxidized form is
also more resistant to unfolding than the reduced species
Gel filtration experiments also corroborate these results
(Table 1) In other words, the following inequality is
maintained: Cu(II)Rc > Cu(I)Rc >> apoRc, where the
symbol larger than means more resistant to unfolding
This behaviour is similar to that reported previously for
Az [22,23,25,53,54] For this BCP this sequence has been
explained by assuming that the metal ion is coordinated in
the unfolded forms According to these studies, the high
stability of Cu(II)Az against the unfolding process is due to
different affinities of the copper(II) ion for the apoform in
the folded and the unfolded states Copper(II) displays
a large degree of affinity for apoAz in the folded state
(DG¼)77.6 kJÆmol)1, pH 7, 20C), while in the unfolded
state the affinity is reduced (DG¼)54.6 kJÆmol)1, same
conditions) [53] The difference clearly favours the Cu(II)Az
folded form A similar, although less marked effect operates
for Cu(I)Az, and obviously, is not present in the case of the
apo form In our study with Rc we did not detect any
evidence of the existence of copper bound to the protein in
the unfolded states However, it is likely that, having a
similar coordination sphere with almost the same kind of
ligands in the unfolded state, the affinity of the copper
should be analogous in this state in both Rc and Az Thus, it
is also probable that the same phenomenon occurs for both
proteins
Relevance in the folding biological process
Unfolding studies are crucial for understanding the folding
process that takes place inside the cell in vivo Rc, unlike
other BCPs, forms aggregates in its unfolding process At
our working concentrations, these intermediates could tend
towards the formation of aggregates If misfolding events do
not occur, it is unlikely that these aggregates will be formed
under biological conditions where the concentration of the
protein is several orders of magnitude lower than those here
used However, keeping in mind that Rc is very stable at low
pH values, this finding could be relevant with regard to the
protein stability in this acid medium It is well-known that many proteins form molten globules at acid pH values [2,55–57], then a possible relationship between the forma-tion of species that are more complex than monomers and stability to acid pH could exist As shown in Fig 5, the hydrophobic residues of Rc all point towards a specific face
of the protein; this could facilitate intermolecular inter-actions Whether or not these interactions are between two homologous proteins (i.e between two proteins of Rc) or with other redox partners, as happens in other BCPs [48–50,58], is still unknown
Aggregates have often been referred to as intermediates prior to the formation of amyloid fibrils in protein (mis)folding [4] It has also been stated that the ability to form amyloid structures is a general feature of polypeptide chains [59] We have observed that the formation/disruption
of Rc aggregates is reversible for a short (2–3 days) period
of time under reducing conditions When no reducing agent
is present, the process is not reversible (probably due to the formation of interchain disulfide bridges) We have also observed the formation of gel-like species in old Rc samples when guanidinium chloride is present They could only be dissolved under strong acid/oxidant conditions Their nature (i.e if they are actually amyloid fibrils) is currently being investigated Thus, when aggregates are present in Rc, they may be prone to form fibrils
Finally, a mention of the role of the metal ion should be made Our results clearly indicate that the copper ion favours Rc folding from the thermodynamic point of view
It follows that the formation of the biologically active (holo) protein consists of two steps: first, the copper binding; and second, the folding process It has been argued that these steps do not occur in this order as the free copper concentration in the cell is exceptionally low (about one molecule of copper per cell [60]) and so, it would be logical for the species with the highest affinity (i.e the folded form)
to take up the copper ion However, kinetic factors are also decisive in this respect In fact, the rate of copper uptake in the folded Az is very low [25,54,61] Rc possesses the most hidden (and the most hydrophobic) copper site of the BCPs (Fig 5) [17,18,45], then it makes sense that this rate is even lower in Rc Thus, it is unlikely that uptake of copper can take place after protein folding The difference observed here regarding the oxidation state is probably not relevant
in vivo, as inside the cell, free copper(II) is toxic and only free copper(I) can be present
Conclusions Unlike other BCPs (such as Pc and Az), Rc forms aggregates (essentially, tetramers and octamers) in the presence of guanidinium chloride This is probably related
to the higher molecular mass of this protein and to its elevated content in hydrophobic residues With regard to the folding process, the holoforms of the protein are more stable than the apoform
Acknowledgements
This work has been supported with financial aid from the DGICYT-Ministerio de Ciencia y Tecnologı´a, Spain (Projects numbers BQU2002-02236 and EET2002-05145) We would like to thank
Trang 8Drs Francisco J Go´mez and Jesu´s M Sanz (both from the Instituto de
Biologı´a Molecular y Celular from the University Miguel Herna´ndez)
for their interesting and helpful comments.
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