Báo cáo khoa học: Unfolding process of rusticyanin Evidence of protein aggregation ppt

Unfolding 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 t echniques. F luorescence and C D spectroscop- ies reveal that the copper ion plays an essential r ole 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 p rotein. Gel filtration c hromatography and glutaraldehyde cross-linking experiments confirm the for- mation of such aggregates. Among t he 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 c ompared with those of other BCPs. Keywords: aggregation; Blue Copper Protein; metalopro- tein; p rotein unfolding; rusticyanin. An understanding of folding p rocesses is crucial in order to determine the causes of protein stability [1–4]. Small proteins typically unfold by m eans of a simple t wo-state mechanism, characterized by the absence of intermediates between the two extreme (folded and unfolded) states. T he process is usually cooperative, reflecting the complementary nature of the tertiary i nteractions t hat ma intain t he p rotein 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 a s 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 o f 16.5 kDa, is the l argest Blue Copper P rotein (BCP) [12,13]. It is also the most abundant protein in Acidithiobacillus ferrooxidans,a Gram negative b acterium t hat ex tract s its e nergy f rom 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 feature s of Rc is its high stability at low pH [16]. Rc possesse s an N-terminal extension (35 amino acids in length), not present in other BCPs, that has been described as a factor protecting the hydrophobic c ore of t he 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 a n i ndependent m odule of the r est o f the protein i n the folded state. Another intriguing property of Rc is i ts redox potential, 680 mV, the highest in t he BCP family [21]. H ow the prote in stabilize s Cu(I) is another que stion that has not been resolved completely. T he efficiency o f 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 f old according to a two-state model. Thus, the kind of question s we address in this study are t he following: (a) are there any intermediate states in the Rc (un)folding proce ss(es) and (b) which oxidation state is preferable for t he folded and unfolded protein? We present here a n exhaustive s tudy 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 metalionanditsoxidationstateareseentobedecisiveinthe 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 coli containing the Rc plasmid [35]. Bacteria cultures were grown in suitably modified M9 medium [20]. Samples (apo, reduced or oxidized Rc) f or all techniques were prepared as described previou sly [20]. Conditions for all experiments (unless otherwise indicated) were acetate Correspondence to A. Donaire, Instituto de Biologı ´ aMoleculary Celular, Universidad M iguel 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 im ages 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 S epte mber 2004, accepted 15 September 2004) Eur. J. Biochem. 271, 4284–4292 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04368.x buffer 10 m M , pH 5.5, 296 K. Eight p ercent of D 2 Owas added to t he sam ples used i n t ranslational d iffusion measurements. In all the titrations and after the a ddition of guanidinium chloride, samples were left for 15 min before making the corresponding measurement. The protein concentration varied according t o the experimental te ch- nique used: 1.2 · 10 )5 M for fluorescence and CD spectros- copies, as w ell as f or cross-linking experiments; a 10-fold concentration (1.2 · 10 )4 M ) for gel filtration chr omato- graphy and ANS fluorescence; and 1.5 · 10 )3 M in diffu- sion NMR experiments. Fluorescence spectroscopy Fluorescence measurements were performed either on a SLM 8000 spectrofluorimeter (Spectronics Instruments, Urbana, I L, USA), interfaced with a Haake w ater bath, or in a Cary Eclipse spectrofluorimeter (Varian, Madrid, Spain), connected to a P elltier cell. A 1.0-cm p ath-length quartz cell (Hellma QS) was used. Changes in t he intrinsic fluorescence of Rc were followed by excitation at 295 nm and its emission spectrum was recorded between 300 and 450 nm. Experiments w ere p erformed with both the apo and the Cu(II) Rc samples. Cu(I) R c t itration 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 t he emission observed a t 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 )1 and the average of four scans was taken. Experiments were performed with the apo or the reduced species, using a cuvette with a path length o f 0.1 cm. After each addition, the values of the ellipticity of the sample between 260 and 2 00 nm were recorded. For each spectrum, the buffer baseline was subt racted. Gel filtration chromatography Analytical gel fi ltration exper iments w ith a po, Cu(I) a nd Cu(II)Rc were performed. Samples were incubated for 30 min i n buffers containing guanidinium chloride at concentrations of 0.0, 1.0, 2.0 or 3.0 M 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 296K. Flow rates of 1mLÆmin )1 were used. For the samples at a 3.0 M guanidinium chloride concentration the flow rate was set to 0.8 mLÆmin )1 to 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 b and 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]: r ¼ ðV e À V 0 Þ V i ð1Þ where, V e is the elution volume of the protein, and V 0 and V i 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 V o and V i volumes were determined using Blue dextran (5 mg ÆmL )1 )and L -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 in verse error function complement of r (erfc )1 [r]), yielding a linear relationship with the molecular Stokes radius, R s [36,37]: R s ¼ a þ b½erfc À1 ðrÞ ð2Þ where, a and b are the calibration constants for the c olumn. The relative volumes of the species corresponding to each band were estimated assuming that they are proportional to R 3 s . According to t he Stokes’ law for a solvated molecule, the translational friction coefficient, f,isgivenbyf ¼ 6pgR s , where, g is the solvent viscosity. The f value for an ideal unsolvated spherical molecule, f 0 , with t he same mass and partial s pecific volume, is given by this expression replacing R s by r 0 . Then, the frictional coefficient of a solvated molecule, f/f 0 , i s given by the ratio R s /r 0 .Foraprotein,r 0 can be c alculated by c onsidering that t he anhydrous molecular volume (M  V =N ) equals to the volume of a sphere: M À  V N ¼ 4 3 pr 0 3 ð3Þ 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, x max , for the hydration in grams of water bound per gram of protein is given b y: x max ¼  V  V water f f 0  3 À 1 "# ð4Þ where  V water isthepartialspecificvolumeofwater (1 c m 3 Æg )1 ). Cross-linking experiments Intermolecular cross-linking experiments [38,39] were per- formed to assess the existence of aggregates. G lutaraldehyde is well-known t o interact with the amine group of Lys residues through its two terminal c arbonyl groups. Thus, it links monomers by means of these amino acids converting them into oligomeric species. Samples of Cu(I)Rc 1.2 · 10 )5 M in the presence of a reducing agent (sodium dithionite) and at guanidinium chloride c oncentrations of 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 M were incubated with Ó FEBS 2004 Unfolding process of rusticyanin (Eur. J. Biochem. 271) 4285 glutaraldehyde 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 a bsence 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 fro m 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 t he ILT program included in t he Bruker XWINNMR package. The intensities of the signals as a function of the gradient strength were fitted to a bi-exponential f unction. Relative hydrodynamic ratios (R h ) b etween the folded a nd unfolded species were compared using the diffusion coefficients and the following expression [43]: R unf h ¼ d fold d unf xR fold h ð5Þ where the d i parameters are the diffusion coefficients, and the s cripts fold and unf refer to the folded and u nfolded species, respectively, present in the solution. The hydrodynamic radius for the folded species were theoretically calculated by using the empirical relationship R h D ¼ 2.21 N 0.57 A ˚ [43], where N indicates t he number of residues. For t he unfolded species, the hydrodynamic radius was estimated by u sing the equation R h N ¼ 4.57 N 0.29 A ˚ . Once these radii were obtained, the compaction factor, C, was calculated from the equation: C ¼ R D h À R h R D h À R N h ð6Þ where R h is the experimental hydrodynamic radius, obtained from the DOSY experiments, according to Eqn (5). The limit values of t he 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 t he apo form, a sigmoid-like b ehaviour in their emission bands is found with a middle point at a guanidinium chloride concentration of 2.1 ± 0.1 M . When the same titration is p erformed with the holoprotein in its oxidized form, no changes in the Trp environments are observed up to concentrations of 5.0 M of the denaturant agent (Fig. 1). At higher concentrations, the unfolding process starts t o take place. The middle point of the v ariation is obtained for a guanidinium chloride concentration ‡ 6.5 M . This value is a l ower limit as the data obtained a t guanidinium chloride 7.2 M (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 s imul- taneously. The parallelism b etween these d ata 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 c ores are exposed to the s olvent 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 displa ys so me CD spectr a of the titration for apoRc. As can be seen, at a high guanidinium chloride concentration (4.0 M ), the spectrum obtained reflects the typical random coil conformation (wi thout any residual secondary structure elements), indicating that the c omplete unfolding process h as already b een completed. F igure 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 % 2.4 M , close to the v alue found b y fluorescence spectroscopy (similar results were obtained with the ellipticity values at 222 nm, data not shown). This probably reflects that the s ame phenomen a a re o bserved with the two techniques. In the same Figure, the t itration of the reduced protein is also shown. A midpoint of 6.3 ± 0.2 M guanidinium Fig. 1. Relative fluorescence e mission of Rc at 351 nm vs. guanidinium chloride concentration. Datacorrespondtotheapo(d), and the oxidized (s)protein. 4286 L. A. Alcaraz and A. Donaire (Eur. J. Biochem. 271) Ó FEBS 2004 chloride 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 w ere not successful. First, n o v ariations were found up to guanidinium chloride concentrations of % 6.0 M . For larger denaturant concentrations, experi- ments were not reproducible, probably due to irreversible reactions between t he copper(II) ion a nd a t hiol atom of the cysteine ligand, as reported previously for azurin [23]. However, as Cu(I)Rc shows measurable c hanges in the CD spectra for concentrations lower than 6.0 M 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 re duced species with regard to the unfolding process. Gel filtration experiments In order to acquire information about the shape, the volume and t he molecular mass o f the folded and unfolded s pecies, gel fi ltration chromatography experiments w ere 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 s mall 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 e lution volumes ( marked with lower cases without and with commas in Fig. 3) appear weakly at guanidinium chloride 1.0 M and are clearly observed at 3 .0 M . The elution volumes of these bands indicate that they procee d f rom species with volumes much larger than that of the monomeric form, i.e. they b elong to aggregates. Analogous results were obtained for the apo and the Cu(II)Rc species. These results are s ummarized 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 d enaturant agent are n eeded. Moreover, at the same concentration of g uanidinium chloride, t he normalized areas o f 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 s pectra of apoRc titra- tion with guanidinium chloride. (B) Relative ellipticity of Rc at 215 nm vs. the gu anidinium chloride concentration. Data corre spond to the apo (d), and the reduced (s)protein. Fig. 3. Gel fi ltration elution bands for Cu(I)Rc. Guanidini um 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. Lowerca se letters with out and with c ommas refer t o bands that correspond to aggregated species. Upper p anel: complete elution filtration. Lower panel: expansion corresponding to band s of aggregated species. *Bands of the void volume o f the column. Ó FEBS 2004 Unfolding process of rusticyanin (Eur. J. Biochem. 271) 4287 The S tokes r adius o f 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 p eaks 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 s ame basis, volumes of approximately eight monomers per molecule are estimated f or bands c¢ and d ¢ (Table 1). T he precise results and the concordance among them for t he three forms of Rc confirm the correctness of the assumed hypothesis put forward (similar volume) and of the conclusion drawn ( existence of tetra and o ctamers). It is noteworthy that the degree of hydration i s low in the folded species, with x max values 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 e nvironment, 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 (x max 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 s pecies independent of the existence and oxidation state of the metal ion. Intermolecular cross-linking Figure 4 s hows t he 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 4.0 M . 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, t he band corresponding to the dimer is attenuated and those o f the monomer and tetramer increase their i ntensity. Therefore, this technique also reveals the existence of aggregates, as observed by gel filtration experiments. Table 1. Peaks observed in gel filtration experiments. The experimen ts w ere pe rformed a t d ifferent co ncentration s o f gu anidinium chloride for the apo, the C u(I) an d the Cu (II) Rc. The denomination o f t he elu tion b ands is the same as i n t he capt ion t o F ig. 3. R S values were o btained b y a pplying 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 expe riments according to Eqn (6). Band [Guanidinium chloride] (M) V e (mL) R S (A ˚ ) x max V rel A rel (%) C factor ApoRc A 0.0 12.7 18.3 0.21 1.00 97.4 1.0 C 2.0 11.6 22.6 1.05 1.00 66.8 0.79 c 2.0 9.3 34.3 3.5 a 24.2 c¢ 2.0 8.3 45.0 7.9 a 9.0 D 3.0 11.3 23.7 1.31 1.00 76.2 0.74 d 3.0 9.07 36.8 3.7 d 8.9 d ¢ 3.0 8.1 48.0 8.3 d 15.0 Cu(I)Rc A 0.0 12.7 18.3 0.21 1.00 98.0 1.0 B 1.0 11.9 21.3 0.75 1.00 97.6 0.86 b 1.0 10.1 29.8 2.7 2.3 C 2.0 11.4 23.2 1.18 1.00 94.1 0.77 c 2.0 9.2 35.4 3.5 4.0 c¢ 2.0 8.2 46.9 8.3 1.9 D 3.0 11.2 24.3 1.46 1.00 73.8 0.72 d 3.0 9.0 36.3 3.3 7.8 d ¢ 3.0 8.0 49.9 8.7 14.1 Cu(II) Rc A 0.0 12.5 19.1 0.34 1.00 95.1 1.0 B 1.0 11.9 21.3 0.75 1.00 97.6 0.89 b 1.0 10.2 28.9 2.5 2.40 C 2.0 11.5 23.2 1.18 1.00 96.4 0.80 c 2.0 9.3 35.2 3.5 2.6 c¢ 2.0 8.3 45.0 7.3 1.1 D 3.0 11.2 24.0 1.39 1.00 85.4 0.76 d 3.0 9.0 36.5 3.5 8.3 d ¢ 3.0 8.0 50.2 9.1 8.3 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. 4288 L. A. Alcaraz and A. Donaire (Eur. J. Biochem. 271) Ó FEBS 2004 Translational diffusion measurements When DOSY measurements were performed in an apoRc sample containing guanidinium chloride at 2.1 M (data not shown), only t wo species (in slow e xchange regime) were distinguished. They both belong to monomer species as the observation of aggregates is precluded b y fast transversal relaxation. The ratio between the diffusion coefficients for these two species was 1.9. Assuming an R fold h value of 18.3 A ˚ for the Cu(I)Rc folded protein (Table 1), a n R unf 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 e ffect, and to a change in the shape of the totally unfolded sp ecies. Using the empirical relationships described in t he Experi- mental procedures [43], r adii of 20.5 a nd 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 t hat t he sp e- 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 existen ce of protein aggregation i n the presence of guanidinium chloride. Gel fi ltration experiments show that the aggregated species contain four or eight mono mers per molecule (Table 1). Cross-linking experiments also reveal the e xistence of aggregates, although, according to this technique, t hey would have h alf 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 o f 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 b e disrupted when SDS is added to t he 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 t he proteins by their redox partners. This situation could be similar f or Rc [51]. It i s 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 d eletion leaves the hydrophobic residues m ore exposed to the solven t. M obility d ata f or 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 r esidues 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 u naltered up to high guanidinium chloride concentrations for t he holoprotein (% 5.0 M ). Thus, if aggregates are b eing formed at lower c oncentra- 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 Ôoff-pathwayÕ 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 re sidues. Blue c olors indicate the position of L ys residues (right side). Coordinates of Rc were obtained from the prote in database (pdb) file 1cur [18]. The green arrow indicates the position of the copper ion (hidd en from th e solvent, insid e the hydropho bic core, left side). T he 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 a n d dotted a rrows ind icate the position of the dimer and tetramer species, resp ectively (see text). Ó FEBS 2004 Unfolding process of rusticyanin (Eur. J. Biochem. 271) 4289 and PsAz [ 33], have shown t hat in these th e 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 B CPs) may p lay a n important role. The existence o f clearly differentiated domains could be one of the factors in the singular behavior of Rc. Second, Rc p ossesses a high content of hydrophobic residues. Moreover, f rom D 2 O/ H 2 O exchange experiments in their folded states, it has been shown that t he 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 t he apoform. Of these, the oxidized f orm is also more resistant to unfolding than the reduced species. Gel fi ltration 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 Ôlarger thanÕ means more resistant to unfolding. This behaviour is similar to that reported previously for Az [22,23,25,53,54]. For t his BCP this sequence has been explained by assuming that the metal ion is coordinated in the unfolded forms. According to these s tudies, t he 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 f olded a nd the unfolded s tates. Copper(II) displays a large degree of affinity for apoAz in the folded state (DG ¼ )77.6 kJÆmol )1 ,pH7,20°C), while in the unfolded state the affinity is reduced ( DG ¼ )54.6 kJÆmo l )1 ,same conditions) [53]. The difference clearly favours t he Cu(II)Az folded form. A similar, although less marked effect operates for Cu(I)Az, and obviously, is not present i n 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 b e 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 t akes place inside the cell in vivo. Rc, unlike other BCPs, forms aggregates in its unfolding process. At our working concentrations, t hese intermediates could tend towards the formation of aggregates. If misfolding events d o 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. H owever, keeping in mind that Rc is very stable at low pH values, this finding could be relevant w ith 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 m onomers and stability t o a cid pH c ould e xist. As shown i n Fig. 5, t he hydrophobic residues of Rc all point towards a specific face of the protein; this could f acilitate intermolecular i nter- 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 t he 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 h ave observed t hat 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 p rocess is not reversible (probably due to the formation of interchain disulfide bridges). We have also observed the formation of gel-like species in old R c samples when guanidinium chloride is present. They could only b e 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 t hermodynamic 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 t he cell is exceptionally low (about one molecule of copper per cell [60]) and so, it w ould be logical for the species with the highest affinity (i.e. t he folded form) to take up the copper ion. However, kinetic factors are also decisive in this respect. I n fact, the r ate o f c opper uptake in the folded A z is v ery low [25,54,61]. R c possesses the Ômost hiddenÕ (and the most hydrophobic) copper site of the BCPs (Fig. 5) [ 17,18,45], then i t makes sense that this rate is even lower in R c. Thus, it is unlikely that uptake of copper c an take place after protein folding. The difference observed here regarding the oxidation state is probably not relevant in viv o, as inside the cell, free copper(II) is toxic and only free copper(I) can be present. Conclusions Unlike other BCPs (such as P c and Az), Rc forms aggregates (essentially, tetramers and octamers) in the presence of guanidinium chloride. This is probably related tothehighermolecularmassofthisproteinandtoits elevated content in h ydrophobic residues. With regar d to the folding process, the holoforms of the protein are more stable than the apoform. Acknowledgements This wo rk has been supported with financial a id from the DGICYT- Ministerio de Ciencia y Tecnologı ´ a, Spain (Projects num bers BQU2002-02236 and EET2002-05145). We would like to thank 4290 L. A. Alcaraz and A. Donaire (Eur. J. Biochem. 271) Ó FEBS 2004 Drs Francisco J . Go ´ mez and Je su´ s M. 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We present here a n exhaustive s tudy of the unfolding process of Rc. Titrations of this protein (in its apo, reduced and oxidized forms)