Báo cáo khoa học: Physico-chemical properties of molten dimer ascorbate oxidase doc

Physico-chemical properties of molten dimer ascorbate oxidase Eleonora Nicolai 1,2 , Almerinda Di Venere 1,2 , Nicola Rosato 1,2 , Antonello Rossi 2 , Alessandro Finazzi Agro’ 2 and Giampiero Mei 1,2 1 INFM, University of Rome, ‘Tor Vergata’, Italy 2 Department of Experimental Medicine and Biochemical Sciences, University of Rome, ‘Tor Vergata’, Italy Dimeric enzymes are largely diffused in living organ- isms, being present in prokaryotes and eukaryotes, and in plants as well as in animals [1,2]. Their func- tionality is strictly dependent on quaternary interac- tions that regulate the stability of subunit interface, as demonstrated by the loss of biological activity often occurring when the two monomers fall apart. Studying the folding process of dimeric proteins is important for several reasons [3]. In fact, their qua- ternary structure represents the simplest case of pro- tein–protein interaction, i.e., the basic mechanism that drives and controls most metabolic pathways in cells [1]. Furthermore, it is the common opinion that oligomers evolved from simpler ancestral structures, namely monomeric proteins whose internal domains might have undergone a swapping process, transform- ing intrachain contacts in a new dimeric interchain surface [4]. Thus, learning the ‘story’ of folding of dimeric (and oligomeric) enzymes is also important to understand part of life evolution. Keywords conformational change; dimeric intermediate; high pressure; protein compressibility; protein folding Correspondence G. Mei, Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Via Montpellier 1, 00133 Rome, Italy Fax: +39 06 72596468 Tel: +39 06 72596460 E-mail: mei@med.uniroma2.it (Received 9 August 2006, revised 26 Sep- tember 2006, accepted 27 September 2006) doi:10.1111/j.1742-4658.2006.05515.x The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that dis- plays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associ- ated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 · 10 8 Pa, providing evidence that mechanically or chemically induced unfolding lead to a simi- lar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DV % )200 mLÆmol )1 ) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Further- more, the combination of guanidine and high pressure uncovers the pres- ence of a marginally stable (DG % 2 kcalÆmol )1 ) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further sup- porting the important role of quaternary interactions in the folding strategy of large dimeric enzymes. Abbreviations AAO, ascorbate oxidase; ANS, 1-anilino-8-naphthalene-sulfonic acid; GdmHCl, guanidinium hydrochloride; MD, molten dimer. 5194 FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS About 10 years ago, the extensive study of small globular protein denaturation allowed a description of the folding process in terms of sound schemes [5–7], from the hydrophobic collapse (typical of molten glob- ule states) to the so called framework model (based on early secondary structure interactions). The discovery of multiple intermediate states in several protein fold- ing experiments, the presence of nearly independently structured domains and the existence of parallel fold- ing pathways led to the hypothesis that these simplified models could actually reflect different aspects of a much more complex process, whose features may be explored by a combination of different experimental tests [8]. In particular, they might be viewed as extremes of a more general nucleation-condensation mechanism, dependent on the balance between early secondary and tertiary interactions [9]. This hypothesis, that has been proposed to hold also in the case of lar- ger enzymes [9], was successfully tested so far only for small proteins where a molecular dynamics simulation is possible [9–11]. In parallel to these experimental approaches, the development of a statistical mechanics theory of the protein folding problem is opening new perspectives in protein folding research [12]. According to this viewpoint, the search for the most stable poly- peptide chain conformation occurs through a rough funnel-shaped energy surface, whose local minima rep- resent possible intermediate species [13]. All these new concepts can be in principle extended also to dimeric proteins. However, the characterization of their folding kinetics and the determination of inter- mediate states is often quite a difficult task, because oligomerization can compete and thus interfere with the assembling of monomers. The analysis of the avail- able crystallographic structures has shown that in the majority of dimeric proteins the intersubunits contact region is quite large with respect to the overall protein surface [2]. Generally the interface is mostly character- ized by hydrophobic side chains that shield each other from water upon dimerization [2]. This feature is very common, but exceptions are found especially in larger dimers, like ascorbate oxidase (AAO; EC 1.10.3.3). This enzyme is a blue copper protein that catalyzes the oxidation of ascorbate with concomitant oxygen reduction to water. It is a homodimer, each subunit containing 14 tryptophans [14] distributed in three dis- tinct domains. Only domains 2 and 3 are involved in the monomer–monomer interaction, through the side chains of residues located in b-turns or random coil segments of the polypeptide chain. Thus, none of the amino acids forming a and b structures are present at the interface. The contact area between the subunits shows a relatively low hydrophobic character, because about half of the amino acids involved are polar. Despite these unusual features the AAO dimer with- stands partial denaturation by % 1.5 m guanidinium hydrochloride (GdmHCl) or % 3 m urea. In this condi- tion no larger aggregates are formed, as demonstrated by analytical ultracentrifugation [15]. In fact, a dimeric folding intermediate is rather observed which lacks catalytic activity and shows a partial loss of tertiary structure. These characteristics recall those of molten globule state of small, globular proteins [16], therefore this dimeric intermediate has been defined as a molten dimer (MD) state [15]. The structural properties of this partially unfolded dimer are obviously correlated to the properties of the intersubunit contacts. Thus, in order to get further information on the monomers association and on the role played by quaternary structure in the stability of large sized enzymes such as AAO, we carried out new measurements using both chemical and physical dena- turing agents. In particular, we took advantage of the possibilities offered by the combination of high pres- sure techniques and low concentrations of GdmHCl [17], to perform a detailed analysis of the intermediate states. The results of steady state and dynamic fluores- cence measurements allowed us to evaluate the volume and free energy changes from the native to the MD state. Further transition to the fully unfolded state has been also shown to be a complex process, involving at least one weak, unstructured monomeric intermediate. These findings provide new insights on the native structure in solution as well as further details on the unfolding pathway that should reflect the mechanism of the protein assembly. Results From native to MD state ) denaturant-induced conformational changes The structural changes of AAO upon partial unfolding by 1.4 m GdmHCl have been investigated by fluores- cence measurements. The intrinsic steady state emis- sion spectra of partially unfolded AAO by 1.4 m GdmHCl or 2.8 m urea are indicative of a greater hydration with respect to the native state (Fig. 1A). In fact a larger full width at half maximum, due to the appearance of a red-shifted shoulder at % 344 nm typ- ical of partially solvent-exposed tryptophan residues, was observed. Lifetime measurements confirm this observation. In fact, in a previous study [14] we repor- ted the rather complex fluorescence decay of AAO, which requires at least two continuous distributions of lifetimes to fit the data. The faster and slower E. Nicolai et al. Probing ascorbate oxidase unfolding routes FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS 5195 distributions were attributed to buried and solvent- exposed tryptophan residues, respectively [14]. As shown in Fig. 1B, there is an increase of the compo- nent at % 2.5 ns in the presence of 1.4 m GdmHCl or 2.8 m urea, while the shorter lifetime component is decreased. Unfortunately these fluorescence decays are unsuitable to monitor rotational correlation times of dozens of nanoseconds, as theoretically expected for a large sized enzyme (i.e., ‡ 58 ns, see below). Therefore, AAO has been labeled with an external probe, namely dansylchloride, which allows anisotropy measurements on longer time scales. Figure 2A shows that the steady state anisotropy of the dansylated enzyme increased upon addition of GdmHCl, up to 1.5 m, indicating a slower tumbling of the protein. The steady state anisotropy of AAO-bound dansylchloride has there- fore been used to get preliminary estimations of the protein volume under native and partially unfolded states. These values, reported in Table 1, have been obtained from the slopes of the linear Perrin plots [18] shown in Fig. 2B. The volume calculated for native AAO is reasonably close to that estimated using the protein molecular mass and an average hydrated speci- fic volume of % 1cm 3 Æg )1 [19]. Instead the volume of the protein molecule at 1.4 m GdmHCl, i.e., the MD species is % 1.8 times larger (Table 1), indicating a rele- vant loss of the original stiffness. These results have been quantitatively confirmed by dynamic anisotropy measurements, in which both the dynamics of the probe and of the whole protein can be determined through their rotational correlation times [18]. As shown in Table 2, a double exponential fit was needed to adequately fit the data. In particular, the longer cor- Fig. 1. (A) Normalized steady state emission spectra, at 10 5 Pa, of AAO at pH 6 (solid line), AAO + 1.4 M GdmHCl (short-dashed line), AAO + 2.8 M urea (dotted line) and AAO + 3.5 M GdmHCl (long dashes). The spectrum at 3 · 10 8 Pa has been reported for com- parison as d (pH 6.0) and h (pH 8). Inset: relative fluorescence total intensity of AAO versus pressure at two different protein con- centration, namely % 10 )7 M (n) and % 10 )5 M (m). (B) Fluorescence lifetime distribution profiles of native AAO, AAO + 1.4 M GdmHCl, AAO + 2.8 M urea, AAO + 3.5 M GdmHCl and AAO at 3 · 10 8 Pa [symbols as in (A)]. Fig. 2. (A) Steady state anisotropy of dansylated AAO as a function of GdmHCl concentration. (B) Perrin plots of the native dansylated enzyme in the absence (s) or in presence of 1.4 M GdmHCl (d). The solid and dashed lines correspond to the best linear fit (yielding Y 1 ¼ 309.9 X + 5.12 and Y 2 ¼ 152.5 X + 4.47, respectively). Probing ascorbate oxidase unfolding routes E. Nicolai et al . 5196 FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS relation time obtained for native AAO (/ 2 % 67 ns) matches closely the theoretical value calculated for a prolate ellipsoidal molecule (/ 2 % 68 ns, see Experi- mental procedures) having the 3D dimensions of AAO [20,21]. The MD, instead, showed a longer correlation time (/ 2 % 124 ns), diagnostic of a much slower tumb- ling. Similar results were also obtained by light scatter- ing measurements, which indeed yielded molecular volumes in good agreement with those estimated from dynamic anisotropy data (Table 1). The MD state is more sensitive to trypsin digestion A further evidence of a less compact tridimensional structure of the MD state has been obtained by trypsin digestion. The results obtained after 30 and 60 min of trypsin digestion are illustrated in Fig. 3, for both native and MD AAO. It appears that already after 30 min the partially folded intermediate is cleaved to smaller fragments (Fig. 3, column E), at variance with native AAO (Fig. 3, column C). After a longer incuba- tion (60 min, columns F, G, H) the proteolysis of MD was more pronounced but a partial digestion of the native sample was observed (column F). Pressure effects on the tertiary structure of native AAO The effect of pressure on AAO intrinsic fluorescence is reported in Fig. 1A. At % 3 · 10 8 Pa a red-shift of the spectrum is accompanied by a 15–20% quenching of tryptophan emission (Fig. 1A, inset). The transition was reversible because the native spectrum was obtained by bringing back the sample to room pres- sure (data not shown). No significant difference was obtained at higher protein concentration (Fig. 1A, inset) ruling out the occurrence of subunit dissociation [22]. Both steady state (Fig. 1A) and dynamic fluores- cence data (Fig. 1B) indicate that high pressure pro- motes a partial, pH-dependent loss of tertiary structure (Fig. 1A). These structural changes are generally characterized by the exposure of internal hydrophobic patches as revealed by 1-anilino-8-naphthalene-sulfonic acid (ANS) binding [23,24]. In a previous study [15], we have in fact shown that the ANS fluorescence is much higher in the partially denatured than in native AAO. A similar effect is achieved under pressure in the range 10 5 –3 · 10 8 Pa (Fig. 4A), further supporting the idea that physical denaturation might parallel the results obtained with chemical denaturants [15]. When nor- malized (Fig. 4A, inset), the data display a sigmoidal shape, suggesting that a two-state transition model (N 2 <- -> MD) may be used to get a fit. A similar trend is shown by the tryptophan fluorescence change Table 1. Theoretical and experimental volumes of native AAO and AAO + 1.4 M GdmHCl. Theoretical dry volume estimated from crystallo- graphic data; theoretical hydrated volume estimated using a hydrated specific volume of 1 cm 3 Æg )1 [19]. Sample Theoretical dry volume [A ˚ 3 ] Theoretical hydrated volume [A ˚ 3 ] Perrin volume [A ˚ 3 ] Dynamic anisotropy volume [A ˚ 3 ] Light scattering volume [A ˚ 3 ] Native 1.74 · 10 5 2.33 · 10 5 2.2 ± 0.3 · 10 5 2.7 ± 0.2 · 10 5 2.4 ± 0.6 · 10 5 Molten dimer – – 4.0 ± 0.3 · 10 5 4.7 ± 0.4 · 10 5 5.2 ± 1.2 · 10 5 Table 2. Rotational correlation times of native AAO and AAO + 1.4 M GdmHCl. Sample v 2 / 1 (ns) / 2 (ns) F 1 (%) AAO (1 exponential) 140 16 ± 8 – 1.00 AAO (2 exponentials) 1.3 0.74 ± 0.05 67 ± 6 0.39 ± 0.02 Molten dimer (1 exponential) 84 43 ± 7 – 1.00 Molten dimer (2 exponentials) 0.9 0.71 ± 0.06 124 ± 9 0.37 ± 0.03 AB C D E F GH Fig. 3. SDS ⁄ PAGE of AAO incubated with trypsin. Columns A and B show the markers and the native AAO, respectively. Patterns C, D and E correspond to AAO, AAO + 1.4 M urea and AAO + 2.8 M urea, incubated with trypsin at 37 °C for 30 min. Patterns F, G and H represent the same runs after 60 min incubation with trypsin. E. Nicolai et al. Probing ascorbate oxidase unfolding routes FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS 5197 as a function of hydrostatic pressure (Fig. 4B). The best fit parameters, representing the free energy and volume changes of the transition, are reported in Table 3. The average DG° value (% 2.9 kcalÆmol )1 )is closer than that previously obtained for GdmHCl- and urea-induced denaturation (% 3.4 kcalÆmol )1 ) in the transition from the native to the MD state [15]. On the other hand, the small, negative volume change, DV 1 ¼ )60 ± 4 mLÆmol )1 (Table 3), is consistent with that observed for many proteins undergoing partial dena- turation under pressure [25]. Denaturant- and pressure-induced effects on the bound copper Copper ions are fundamental for the AAO redox activ- ity. The enzyme contains four metal ions per subunit classified as mononuclear (type I Cu) and tri-nuclear (type II and type III Cu) copper centers, respectively [20]. They are characterized by distinct optical proper- ties with absorption peaks at 610 (type I) and 330 nm (type II–III) [26]. The absence of activity in the MD state [15] indicates that even a partial loss of tertiary structure has strong effects on copper. We have there- fore investigated the copper sites by absorption spec- troscopy (Fig. 5). Figure 5A demonstrates that the tri-nuclear copper center structure is already lost under quite mild unfolding denaturant concentrations or pressure values. On the contrary, the type I Cu binding site is only partially affected by the addition of 1.4 m GdmHCl and, to a lesser extent, by high pressure, but only at pH 8 (Fig. 5B). Pressure-induced dissociation and unfolding of the MD species A next set of experiments concerned the combined effect of GdmHCl (1.4 m) and pressure on AAO. The fluorescence of AAO-bound ANS decreased at increasing pressure indicating a gradual dissociation of the probe from the protein, due to the collapse of the enzyme tridimensional scaffolding (Fig. 6A). This result was confirmed by intrinsic fluorescence meas- urements as shown in Fig. 6B. The center of mass of the steady state fluorescence spectrum was progres- sively shifted to longer wavelengths upon increasing Fig. 4. (A) Pressure-induced ANS binding to AAO. The arrow indi- cates the dependence of the ANS fluorescence intensity upon increasing the hydrostatic pressure up to 3 · 10 8 Pa (ANS ⁄ protein % 10 : 1). The inset shows the ANS fluorescence intensity as a function of pressure. The best fitting parameters obtained by a two-state fit (N 2 <–> MD) are reported in Table 3. (B) Effect of high pressure the fluorescence center of mass of AAO at pH 6.0 (d) and pH 8.0 (h ). Solid lines represent a fit for two-state transition (N 2 <–> MD) and the corresponding parameters are reported in Table 3. Table 3. Results of native to MD (N2 <–> MD) and MD to unfolded (MD <–> 2M* <–> 2U) pressure-induced transition fits. Transition and fitting model v 2 DG 1 ° [kcalÆmol )1 ] DV 1 [mLÆmol )1 ] DG 2 ° [kcalÆmol )1 ] DV 2 [mLÆmol )1 ] N2 <–> MD ANS pH 6.0 1.1 3.0 ± 0.3 )62 ± 5 – – N2 <–> MD TRP pH 6.0 0.9 2.8 ± 0.2 )59 ± 3 – – N2 <–> MD TRP pH 8.0 1.0 3.4 ± 0.2 )69 ± 4 – – MD <–> 2M* <–> 2U TRP center of mass 1.1 10.6 ± 0.3 )192 ± 20 2.0 ± 0.1 )54 ± 4 MD <–> 2M* <–> 2U TRP intensity 1.3 10.4 ± 0.9 )161 ± 14 1.8 ± 0.2 )48 ± 5 MD <–> 2M* <–> 2U ANS intensity 1.5 11.1 ± 0.3 )161 ± 12 2.2 ± 0.3 )61 ± 6 Probing ascorbate oxidase unfolding routes E. Nicolai et al . 5198 FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS the hydrostatic pressure. In particular, at 3 · 10 8 Pa the spectrum is almost completely red-shifted, as expected for fully solvated tryptophan residues (Fig. 6B, inset). Both the intrinsic fluorescence inten- sity and spectrum position are much more affected than ANS fluorescence at low pressure values (10 5 – 4 · 10 7 Pa), suggesting that the two fluorophores are probing nonsimultaneous structural transitions. Indeed, at variance with the transition from the native to the MD state, it was impossible to fit the data reported of Fig. 6B as a simple two-state process. More complex fits were therefore attempted, taking into account a further intermediate state. Because the transition is protein-concentration dependent (Fig. 6B, inset) both ANS (Fig. 6A) and tryptophan fluorescence data (Fig. 6B) have been interpreted according to the following scheme: MD < ÀÀ > 2M Ã < ÀÀ > 2U where M* and U represent partially and fully unfol- ded monomers, respectively. The overall free energy change (DG 1 + DG 2 ) calculated from these experi- ments ranges between 12.2 and 13.3 kcalÆmol )1 (Table 3), a value remarkably close to that previously obtained for the transition of MD to the fully unfol- ded state, upon chemical denaturation (urea or GdmHCl [17]). The dissociation is accompanied by a very large volume change (Table 3) and is inhibited by glycerol (Fig. 6B), a known stabilizing agent [27]. Figure 6 demonstrates that at 30% glycerol the trans- ition curve was shifted by % 5 · 10 7 Pa to higher pres- sure, while at 70% glycerol the displacement of the spectral center of mass was hardly more than 1.5 nm, up to 2.5 · 10 8 Pa. The addition of glycerol did not produce detectable changes to the native protein (data not shown). Discussion Early events in the denaturation of AAO ) preferential hydration at the domain interface The characterization of predissociated states plays a crucial role in the study of oligomeric proteins, yield- ing important information on the relationships between tertiary and quaternary interactions. In fact, besides the stabilization of the tridimensional structure, these interactions regulate volume fluctuation and con- formational changes, i.e., those dynamic properties that actually keep the enzymes working. Independent measurements on AAO partially unfolded by GdmHCl, such as dynamic fluorescence, anisotropy and light scattering provide quantitative structural infor- mation along the denaturation pathway (Tables 1–3). In particular, the slower rotational dynamics (/ 2 % 125 ns) of the dimeric intermediate suggests the occur- rence of a swelling effect, probably associated with the progressive hydration of the protein tertiary structure. The pressurization of native AAO in the range 10 5 – 3 · 10 8 Pa also produces a predissociated intermediate 0.006 0.008 0.010 0.012 310 330 350 absorbance wavelength (nm) A 0.000 0.004 0.008 0.012 560 600 640 680 wavelength (nm) B Fig. 5. Dependence of the AAO absorption bands on pressure (10 5 Pa, d;6· 10 7 Pa pH 6, ––; 6 · 10 7 Pa pH 8, —). The spectrum at 10 5 Pa in the presence of 1.4 M GdmHCl or 2.8 M urea has been reported for comparison (h). (A) represents the near UV absorption (type II–III tri-nuclear copper center), while (B) corresponds to visible absorption (type I, blue copper center). E. Nicolai et al. Probing ascorbate oxidase unfolding routes FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS 5199 where the content of residual structure is pH-depend- ent (Figs 1A and 4B). The data indicate that pressure generates an intermediate much alike the denaturant- induced MD state, showing a larger heterogeneity of the emission decay (Fig. 1B) and an enhanced propen- sity to bind ANS (Fig. 4A). The effect of high pressure on proteins’ stability has been extensively studied in the last decade, in order to gain new insight into its peculiar denaturation mechan- ism. The protein’s interior is known to behave as a solid particle, its compressibility being generally very low [28–30]. In the case of monomeric globular pro- teins, pressure unfolding has therefore been associated with a partial hydration of the peripheral protein regions, in the presence of a still and stiff hydrophobic core [30,31]. On the other hand, the behavior of large proteins is more complex. For instance, dimeric enzymes display both elastic and anelastic changes prior to subunit dissociation [32]. Moreover, large pro- teins gain a greater flexibility through the reciprocal movements of their domains [33], suggesting that their weak points with respect to both physical and chemical stress are mainly located in their interdomain regions rather than inside each individual domain. The X-ray structure of AAO revealed the existence of three dis- tinct domains per subunit [20,21], sharing a common b-barrel topology. This particular scaffolding can indeed account for the protein resistance to high pres- sure in the range 10 5 –3 · 10 8 Pa. Beta structures show a lower compressibility than loops and a-helical regions [34]. Furthermore, they are also the most appropriate shelters for hydrophobic residues from water, according to a recent report on the accessible surface of about 600 protein structures [35]. Even though volume changes upon protein unfold- ing may arise from different sources [36], the so called electrostriction effect seems to play a major role in the pressure-induced MD state of AAO [25,29,30,37]. Such a hypothesis is not only supported by the strong pH-dependence observed in the first pressure-induced transition, but also by the presence of an uncommonly large number [46] of ion pair interactions [21], 13 of which occur between different domains of the same subunit. This finding and the relapse of the tri-nuclear copper site (located between domains 2 and 3) at only 6 · 10 7 Pa (Fig. 5A) are further evidence that the swelling effect from the native to the MD state arises from the preferential hydration of interdomain sites, rather than of the domains’ core. In this model, the hydration of the interdomain regions would recall the mechanism that characterizes the early denaturation steps of the peripheral shell of smaller monomeric pro- teins. This is in keeping with the overall similarity of the three domains in AAO each of which mimics the small blue copper proteins [20,21]. The reversibility principle would require that the formation of interdo- main contacts within the same subunit was a very late event of the folding process. It is important to recall that in all copper proteins studied the metal is not A B Fig. 6. (A) Pressure-induced ANS dissociation from the AAO MD intermediate (at 1.4 M GdmHCl). The arrow indicates the depend- ence of the ANS fluorescence intensity upon increasing the exter- nal hydrostatic pressure from 10 5 –3 · 10 8 Pa. The inset shows the ANS fluorescence intensity as a function of pressure. The best fit- ting parameters (Table 3) were obtained by a three-state fit (MD <–> 2 M* <–> 2U). (B) Effect of high pressure and glycerol on the intrinsic fluorescence of the MD intermediate. Filled symbols repre- sent the spectral center of mass in the absence (circles), or pres- ence of 30% (diamonds) and 70% (triangles) glycerol. Empty circles correspond to the change of the total fluorescence intensity in the absence of glycerol. The three state (MD <–> 2M* <–> 2U) fits of the tryptophan center of mass and total intensity as a func- tion of pressure are reported as solid lines. The results of the two fits are shown in Table 3. The inset shows the spectra of the MD state at 1.8 · 10 8 Pa at two different AAO concentrations, namely 6 l M (solid line) and 0.2 lM (short dashes). The spectrum at % 3 · 10 8 Pa (0.2 lM, dotted line) and the spectrum of the fully unfolded AAO (10 5 Pa in the presence of 3.5 M GdmHCl, long dashes) are also reported for comparison. Probing ascorbate oxidase unfolding routes E. Nicolai et al . 5200 FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS required for folding. Furthermore it is possible to obtain partially or fully copper-depleted AAO without significantly affecting the structure. The role of dimerization in the folding strategy of a large protein The combination of low denaturant concentration and pressure opens new possibilities to the study of protein folding [17], allowing us to trap intermediates charac- terized by a smaller volume than that of the native state, undetectable with traditional denaturation tech- niques. The ‘softer’ action of hydrostatic pressure has also been proposed to increase the local roughness of the folding energy landscape [38], depending on the protein’s structural features. Our results are in keeping with these ideas. In fact, no stable monomeric species has been previously observed upon urea or GdmHCl denaturation of AAO [15]. However, the analysis of the GdmHCl + pressure unfolding curves (Fig. 6A,B and Table 3) demonstrates that monomeric species might be formed when the MD state at 1.4 m GdmHCl is pressurized. The addition of glycerol, which is known to reduce the size of internal cavities [27], coun- teracts the loss of the protein quaternary structure (Fig. 6B). Therefore, at variance with the electrostric- tion mechanism, which might explain the volume chan- ges from the native to the MD states, the collapse of the voids at the dimeric interface might be the main reason for the unusually large negative DV value (% )200 mLÆmol )1 ) observed upon pressure-induced dissociation. Indeed, the visual inspection of the AAO structure demonstrates that a large gap exists between the two subunits (Fig. 7A). A more quantitative calcu- lation of the AAO gap volume index, i.e., the ratio between the gap volume and the interface area [39,40], shows that it is by far the largest value obtained among the available crystallographic structures of dimeric proteins (Fig. 7B). This is due to both the gap volume and the very small size of the contact interface, which in fact involves only 25 amino acid side chains per subunit. Interestingly, these residues are not ran- domly distributed along the two polypeptide chains, but rather form 3–4 stretches of neighbor, if not adja- cent, residues. This arrangement would suggest for AAO a very early quaternary interaction between a few patches of neighbor residues, probably in order to stabilize only partially folded monomeric species. A recent study of a small homodimeric protein, namely the factor for inversion stimulation, has demonstrated that the formation of a dimeric intermediate can indeed be a strategy to accelerate the folding process [41]. Unfortunately detailed kinetic folding studies on larger oligomeric proteins are still at the early stages, but there are examples in which a fast subunit associ- ation occurs before the final folding state is achieved [42]. On the other hand, if the folding of the AAO mo- nomers is strictly intertwined with the formation of the quaternary interactions, one might expect to find clues of this process in the same folded protein. For instance, an early dimerization followed by a collapse toward a more compact structure might lead to larger inclusion of solvent molecules within the protein mat- rix. The crystallographic model of AAO reported in Fig. 7A supports this hypothesis. In fact it appears that several water molecules are entrapped in cavities not accessible from the outside. More interestingly, their distribution is uneven, with the area near the dimeric interface being crowded by water molecules. B A Fig. 7. (Top) Crystallographic model of dimeric AAO (PDB file 1AOZ) obtained by removing all water molecules at the protein sur- face. The AAO structure has been reported in the transparence mode, in order to show the water molecules fully entrapped within the protein matrix (purple). (Bottom) Gap volume index (i.e., the ratio between the total empty gap volume at the dimeric interface and the dimeric interface area) of % 30 dimeric proteins as a func- tion of their size (the black bar corresponds to AAO). E. Nicolai et al. Probing ascorbate oxidase unfolding routes FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS 5201 Conclusion Water plays a major, dual role in proteins’ life [43,44]. The final amount of water molecules entrapped within a protein structure arises as the best compromise among several requirements, namely folding, stability and functionality. In the case of enzymes characterized by a quaternary structure these features are particularly critical to understand the reason for oligomerization, when no simple explanation (e.g., allosteric effects) hold [2,3]. The analysis of specific cases, such as the folding process of AAO, can be therefore paradigmatic for other large dimers. The stabilization of the partially folded monomers through nonlocal interactions within each subunit’s tertiary structure is entropically costly, as it would drastically reduce the degrees of freedom of the polypeptide chain [45]. We propose that quaternary interactions are fundamental to stabilize each subunit in AAO and to drive the final folding from a hydrated MD state to the native conformation. Although a gen- eralization is premature, it will be interesting to see whether, and to what extent, the stabilization through similar intermediates is a potential, more general trick to drive (and probably accelerate) the overall assembly process, thereby influencing the balance between enthal- pic and entropic contributions. After all, such a mech- anism would also avoid exceedingly high stabilization energy barriers for the folding of large dimers at the expense of water inclusion in the final structure. The relatively low stabilization energy of large molecules could be a natural, built-in advantage that might also allow a more rapid turnover of these proteins in vivo. Experimental procedures Materials Ultrapure GdmHCl and urea were purchased from USB (United States Biochemicals; Cleveland, OH, USA); dansyl- chloride and ANS were purchased from Sigma (St Louis, MO, USA). Ascorbate oxidase from green zucchini was purchased from Boehringer Mannheim (Mannheim, Germany) and dissolved in 80 mm potassium phosphate buffer, pH 6, at 20 °C, unless otherwise specified. Protein dansylation AAO in potassium-phosphate buffer (pH 8) was incuba- ted, at 20 °C, in the presence of dansylchloride (ratio % 1 : 30). After 24 h, the dansylchloride in excess was removed, by filtration of the solution through a D-salt ex- cellulose plastic desalting column (exclusion limit M r 5000, equilibrated at pH 6), thus isolating the dansylated protein fractions. Spectroscopic assays Steady state fluorescence spectra and anisotropy were recor- ded on a photon counting spectrofluorometer equipped with Glan Thompson polarizers (ISS, model K2, Champaign, IL). Dynamic fluorescence measurements were carried out using the phase-shift and demodulation technique [18]. The light sources of the dynamic fluorometer (ISS, model Koala) were either a K180 laser diode, with emission wavelength at 370 ± 8 nm or an arc-xenon lamp modulated in the range 2– 200 MHz. The data analysis was performed using the glo- bals unlimited software (LFD, Urbana, IL, USA) [46]. Steady state and dynamic fluorescence at high pressure were measured with the same instruments, using the high pressure ISS cell, equipped with an external bath circulator. Light scattering measurements were performed on a Horiba (Kyoto, Japan) LB-500 dynamic light scattering nanoparticle size analyzer, equipped with a 650 nm, 5 mW laser diode. Data analysis was performed using the accompanying soft- ware based on a Fourier-transform deconvolution procedure. Absorption spectra in the range % 300–700 nm were recorded connecting the pressure cell to a PerkinElmer (Wellesley, MA, USA) Lambda 18 spectrometer, through a couple of Hellma (041.002-UVS) fiber optic cables (Hellma, Milan, Italy). Theoretical estimation of the average rotational correlation time A preliminary, rough estimation of the overall rotational correlation time for a spherical molecule with the same size of AAO has been obtained using the following approxima- ted relationship [19]: U sph ¼ 1=6D rot %ðM r =2:4Þ10 À12 s % 58 ns where D rot and M r represent the rotational diffusion coeffi- cient and the protein molecular mass (140 000 Da), respect- ively. A more reliable value has been instead obtained considering that the AAO dimer is a prolate ellipsoid with semiaxes a % 110 A ˚ and b % 55 A ˚ [20], yielding the follow- ing relative rotational correlation values [19]: U a =U sph ¼ 1:51 U b =U sph ¼ 1:05 i.e., F a % 88 ns and F b % 61 ns. The harmonic mean of these values (i.e., the parameter obtained by anisotropy dynamic measurements) is therefore: <U>% 68 ns Molecular graphics The model of dimeric AAO was obtained from the PDB file 1AOZ [20,21] and elaborated with the visual molecular dynamics (vmd) software (Urbana, IL, USA). The analysis Probing ascorbate oxidase unfolding routes E. Nicolai et al . 5202 FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS of the dimeric interface of AAO was carried out using the ‘Protein–Protein’ Interaction Server (http://www.biochem. ucl.ac.uk/bsm/PP/server/ [39,40]). References 1 Marianayagam NJ, Sunde M & Matthews JM (2004) The power of two: protein dimerization in biology. Trends Biochem Sci 29, 618–625. 2 Mei G, Di Venere A, Rosato N & Finazzi Agro’ A (2005) The importance of being dimeric. Febs J 272, 16–27. 3 Ali MH & Imperiali B (2005) Protein oligomerization: how and why. Bioorg Med Chem 13, 5013–5020. 4 Jaenicke R & Lilie H (2000) Folding and association of oligomeric and multimeric proteins. Adv Prot Chem 53, 329–401. 5 Brockwell DJ, Smith DA & Radford SE (2000) Protein folding mechanisms: new methods and emerging ideas. Curr Opin Struct Biol 10 , 16–25. 6 Radford SE (2000) Protein folding: progress made and promises ahead. Trends Biochem Sci 25, 611–618. 7 Fersht AR & Daggett V (2002) Protein folding and unfolding at atomic resolution. Cell 108, 573–582. 8 Wallace LA & Matthews CR (2002) Sequential vs. par- allel protein-folding mechanism: experimental tests for complex folding reactions. Biophys Chem 101–102, 113– 131. 9 Daggett V & Fersht AR (2003) Is there a unifying mechanism for protein folding? Trends Biochem Sci 28, 18–25. 10 Gianni S, Guydosh NR, Khan F, Caldas TD, Mayor U, White GWN, De Marco ML, Daggett V & Fersht AR (2003) Unifying features in protein-folding mechanisms. Proc Natl Acad Sci 100, 13286–13291. 11 White GWN, Gianni S, Grossmann JG, Jemth P, Fersht AR & Daggett V (2005) Simulation and experi- ment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding. J Mol Biol 350, 757–775. 12 Brooks CL III, Onuchic JN & Wales DJ (2001) Taking a walk on a landscape. Science 293, 612–613. 13 Onuchic JN, Schulten ZL & Wolynes PG (1997) The energy landscape perspective. Annu Rev Phys Chem 48, 545–600. 14 Di Venere A, Mei G, Gilardi G, Rosato N, De Matteis F, McKay R, Gratton E & Finazzi Agro’ A (1998) Resolution of the heterogeneous fluorescence in multi- tryptophan proteins: ascorbate oxidase. Eur J Biochem 257, 337–343. 15 Mei G, Di Venere A, Buganza M, Vecchini P, Rosato N & Finazzi Agro’ A (1997) The role of the quaternary structure in the stability of dimeric proteins: the case of ascorbate oxidase. Biochemistry 36, 10917–10922. 16 Kuwajima K (1989) The molten globule state as a clue for understanding the folding and cooperativity of glob- ular-protein structure. Proteins 6, 87–103. 17 Perrett S & Zhou JM (2002) Expanding the pressure technique: insights into protein folding from combined use of pressure and chemical denaturants. Biochim Biophys Acta 1595, 210–223. 18 Lakowicz JR (1999) Principles of Fluorescence Spectros- copy, 2nd edn. Kluwer Academic ⁄ Plenum Press, New York, NY. 19 Cantor CR & Schimmel PS (1980) Biophysical Chemis- try, Vol. II. Freeman, San Francisco, CA. 20 Messerschmidt A, Rossi A, Ladenstein R, Huber R, Bolognesi M, Gatti G, Marchesini A, Petruzzelli R & Finazzi Agro’ A (1989) X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. J Mol Biol 206, 513–529. 21 Messerschmidt A, Ladenstein R, Huber R, Bolognesi M, Avigliano L, Petruzzelli R, Rossi A & Finazzi Agro’ A (1992) Refined crystal structure of ascorbate oxidase at 1.9 A ˚ resolution. J Mol Biol 224, 179–205. 22 Silva JL & Weber G (1993) Pressure stability of pro- teins. Annu Rev Phys Chem 50, 245–252. 23 Lakowicz JR (1983) Principles of Fluorescence Spectros- copy. Plenum Press, New York, NY. 24 Semisotnov GV, Rodionova NA, Razgulyaev OI, Uversky VN, Gripas’ AF & Gilmanshin RI (1991) Study of the ‘Molten Globule’ intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31, 119–128. 25 Royer CA (2002) Revisiting volume changes in pres- sure-induced protein unfolding. Biochim Biophys Acta 1595, 201–209. 26 Avigliano L & Finazzi Agro’ A (1997) Biological Function and Enzyme Kinetics of Ascorbate Oxidase. In Multi-Copper Oxidases (Messerschmidt A, ed), pp. 265–278. World Scientific Publishing Co Pte Ltd, Singapore. 27 Priev A, Almagor A, Yedgar S & Gavish B (1996) Gly- cerol decreases the volume and compressibility of pro- tein interior. Biochemistry 35, 2061–2066. 28 Gavish B, Gratton E & Hardy CJ (1983) Adiabatic compressibility of globular proteins. Proc Natl Acad Sci 80, 750–754. 29 Kharakoz DP (2000) Protein compressibility, dynamics and pressure. Biophys J 79, 511–525. 30 Chalikian TV (2003) Volume tric properties of proteins. Annu Rev Biophys Biomol Struct 32, 207–235. 31 Taulier N & Chalikian TV (2002) Compressibility of protein transitions. Biochim Biophys Acta 1595, 48–70. 32 Cioni P & Strambini GB (1996) Pressure effects on the structure of oligomeric proteins prior to subunit disso- ciation. J Mol Biol 263, 789–799. E. Nicolai et al. Probing ascorbate oxidase unfolding routes FEBS Journal 273 (2006) 5194–5204 ª 2006 The Authors Journal compilation ª 2006 FEBS 5203 [...]... 39 Jones S & Thornton JM (1995) Protein–protein interactions: a review of protein dimer structures Prog Biophys Mol Biol 63, 31–65 5204 40 Jones S & Thornton JM (1996) Principles of protein–protein interactions Proc Natl Acad Sci 93, 13–20 41 Topping TB, Duane A, Hoch DA & Gloss LM (2004) Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation J Mol Biol 335, 1065–1081 42... chemical shift in BPTI Protein Sci 8, 1946–1953 35 Lins L, Thomas A & Brasseur R (2003) Analysis of accessible surface of residues in proteins Protein Sci 12, 1406–1417 36 Harpaz Y, Gerstein M & Chotia C (1994) Volume changes on protein folding Structure 2, 641–649 37 Royer CA (2005) Insights into the role of hydration in protein structure and stability obtained through hydrostatic pressure studies Braz...Probing ascorbate oxidase unfolding routes E Nicolai et al 33 Gerstein M, Lesk AM & Chothia C (1994) Structural mechanism for domain movements in proteins Biochemistry 33, 6739–6749 34 Akasaka K, Li H, Yamada H, Li R, Thoresen T & Woodwar CK (1999) Pressure response of protein backbone structure Pressure-induced amide15N chemical shift... & Gloss LM (2004) Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation J Mol Biol 335, 1065–1081 42 Doyle SM, Bilsel O & Teschke CM (2004) SecA folding kinetics: a large dimeric protein rapidly forms multiple native states J Mol Biol 341, 199–214 43 Ball P (2003) How to keep dry in water Nature 423, 25–26 44 Pace CN, Trevino S, Prabhakaram E & Scholtz JM ˜ (2004) Protein . therefore this dimeric intermediate has been defined as a molten dimer (MD) state [15]. The structural properties of this partially unfolded dimer are obviously correlated to the properties of the intersubunit. presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric. Physico-chemical properties of molten dimer ascorbate oxidase Eleonora Nicolai 1,2 , Almerinda Di Venere 1,2 , Nicola Rosato 1,2 ,