Tài liệu Báo cáo khoa học: Evidence for noncooperative metal binding to the a domain of human metallothionein ppt

Evidence for noncooperative metal binding to the a domain of human metallothionein Kelly E. Rigby Duncan and Martin J. Stillman Department of Chemistry, The University of Western Ontario, London, ON, Canada Over the past several decades, significant advances have been made in the field of protein folding [1–4]. However, the direct and specific involvement of metal ions in the folding process of metalloproteins has received far less attention, despite the fact that one- third of all known enzymes require metal ions for structural or functional purposes [5]. Post-translational metal-induced protein folding is a vital process that still requires mechanistic elucidation. Metalloproteins that bind multiple metals introduce an additional layer of complexity, in that cooperative metal-binding mech- anisms are possible in which the complete multiple metal-binding site forms in preference to partially filled binding sites. Metallothionein (MT) is a metalloprotein found in nearly all mammalian tissues coordinated to multiple group 11 and 12 metal ions [6]. The high capacity of MT to bind both essential and nonessential metal ions in vivo and in vitro strongly suggests a role in metal ion storage, metabolism and trafficking of Cu and Zn, as well as sequestration of Cd and Hg; however, the exact function of MT remains undefined. More recently, MT has been implicated in brain tissue repair through anti-inflammatory, antioxidant and antiapop- totic roles [7–11], as well as in chemotherapy resistance [12]. Domain-independent but metal ion-directed fold- ing of MT results in the formation of discrete metal– thiolate clusters within each of the a and b domains with stoichiometries of [M 4 (S cys ) 11 ] and [M 3 (S cys ) 9 ], respectively, for divalent metal ions (M) [13–15]. One of the most biologically important, and contro- versial, questions regarding the metallation of the two MT domains is whether the metal-binding reaction proceeds by a positively cooperative mechanism. The ramification of cooperative metal binding is that only the completely metallated and folded domains would have functional significance. The metal-binding proper- ties of MT have been extensively investigated in the past, primarily as in vitro metallation reactions with different MT isoforms and varying metal ions [16–21]. Although most of these publications are from 10–20 years ago, these reports still represent a common Keywords CD; cooperativity; metal-dependent protein folding; metallothionein; MS Correspondence M. J. Stillman, Department of Chemistry, Chemistry Building, The University of Western Ontario, London, ON, Canada, N6A 5B7 Fax: +1 519 661 3022 Tel: +1 519 661 3821 E-mail: martin.stillman@uwo.ca Website: http://www.uwo.ca/chem/ (Received 22 December 2006, revised 2 February 2007, accepted 1 March 2007) doi:10.1111/j.1742-4658.2007.05762.x In the present study, we investigated the metal-binding reactivity of the isolated a domain of human metallothionein isoform 1a, with specific emphasis on resolving the debate concerning the cooperative nature of the metal-binding mechanism. The metallation reaction of the metal-free a domain with Cd 2+ was unequivocally shown to proceed by a non- cooperative mechanism at physiologic pH by CD and UV absorption spectroscopy and ESI MS. The data clearly show the presence of interme- diate partially metallated metallothionein species under limiting Cd 2+ con- ditions. Titration with four molar equivalents of Cd 2+ was required for the formation of the Cd 4 a species in 100% abundance. The implications of a noncooperative metal-binding mechanism are that the partially metallated and metal-free species are stable intermediates, and thus may have a poten- tial role in the currently undefined function of metallothionein. Abbreviations a-rhMT -1a, recombinant a domain of human metallothionein isoform 1a; MT, metallothionein. FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS 2253 view of metal binding to MT and have been cited regularly in recent reports. The data presented in these papers clearly show domain-specific binding for M 2+ (M ¼ Zn, Cd) initially to the a domain, thereby lead- ing to formation of the M 4 a cluster prior to formation of the M 3 b cluster, demonstrating that the individual binding constants of divalent metal ions for the a domain are larger than those for the b domain. Further interpretation of these data resulted in the proposal of positively cooperative metal binding as the primary metallation mechanism for each of the two domains [18–21]. Closer inspection of the previously published data, however, brings the claim of positive cooperativity into question, as there is no direct evi- dence to show that coordination of the first metal ion to the a domain enhances the binding of the second metal ion and so forth. Indeed, other published reports present data showing that partially metallated species of Cd–MT or Zn–MT exist under limiting metal ion conditions, suggesting a noncooperative mechanism [22,23]. Recent kinetic results for As 3+ binding to the two isolated domains were also interpreted in terms of a series of noncooperative bimolecular reactions [24]. The fact that Cd 2+ has been shown to coordinate to the two-domain ba-MT in a domain-specific manner, with a preference for the a domain, has been construed as being an indicator of cooperative metal binding to the a domain. This study focuses on the metallation of the isolated a domain, with the purpose of clarifying this point. Additionally, the reported concurrent metal- lation of both domains in the two-domain protein by Co 2+ [25] and Cd 2+ [23] provides an excellent example of the complexity introduced by the presence of the b domain in efforts to elucidate the potentially cooper- ative nature of the metal-binding reaction within each of the domains. Thus, the goal is to elucidate the meta- llation mechanisms of the individual domains, in the hope, initially, of simplifying the interpretation of the metallation details of the two-domain protein. The results presented here allow successful and complete interpretation of the previous data in terms of non- cooperative, domain-specific metal binding. Results Investigation into the metal-binding mechanism of the isolated a domain was carried out on the recombin- antly synthesized a domain fragment of human MT isoform 1a (a-rhMT-1a). The recombinant protein was prepared by overexpression in Escherichia coli as an S-tag fusion protein in the presence of Cd 2+ (see Experimental procedures for a full description of the protein preparation and purification details). Following isolation and purification, the S-tag fusion peptide was cleaved from the domain, generating the isolated a domain, the sequence of which is shown in Fig. 1A. The four divalent metal ions are labeled 1a)4a, and the 11 cysteinyl sulfurs are labeled 1–11, starting from the N-terminus. Figure 1B shows the space-filling and ball-and-stick representations of Cd 4 a-rhMT-1a, emphasizing the wrapping of the polypeptide backbone in a left-handed coil around the metal–thiolate cluster, which is shown in the space-filling model to be located in the center of the domain. Figure 1C shows the iso- lated Cd 4 (S cys ) 11 cluster, where each cadmium ion (green spheres) coordinates tetrahedrally to four cystei- nyl sulfurs (yellow spheres), such that five of the 11 cysteinyl sulfurs act as bridging ligands between two metal centers, and the remaining six act as terminal ligands by coordinating to a single metal center. The numbering of the cadmium ions and the cysteinyl sul- furs in Fig. 1C corresponds with that in the sequence shown in Fig. 1A. Demetallation to produce the metal- free apo-a-rhMT was carried out by eluting the cadmium-containing domain through a size exclusion column equilibrated with a low-pH eluant. The term ‘positive cooperativity’ refers to an increase in equilibrium constant (K) for each step of a sequential reaction; in other words, coordination of the first metal ion facilitates the binding of the second metal ion, and so forth. Experimentally, this translates into the detection of only the initial species, in this case the metal-free protein, and the final species, which is the fully metallated holoprotein, with no detectable intermediate species. Thus, with substoichiometric additions of Cd 2+ to apo-a-rhMT , the metal-free pro- tein will be detected together with a corresponding fraction of the metal-saturated Cd 4 a species if the met- allation mechanism proceeds by a positively cooper- ative pathway. Alternatively, the partially filled Cd 1 a, Cd 2 a and Cd 3 a intermediate species will be detected in the case of a noncooperative metallation mechanism. The metallation rate of either the cooperative or non- cooperative process would depend on the preliminary conformation of the protein and the coordination properties of the incoming metal ions. Metallation of apo-a-rhMT-1a with Cd 2+ was car- ried out at pH 7.3 by raising the pH of the apo-MT solution prior to the addition of the cadmium ions. Previous kinetic data reported by Ejnik et al. [26] showed metallation of MT with Cd 2+ to be complete within the 4 ms mixing time of the stopped-flow instru- ment at room temperature. From this, the metallation of the a domain with Cd 2+ can be considered a nearly instantaneous reaction. In addition, no evidence has been reported to show that any change occurs to the Noncooperative metallation of metallothionein K. E. Rigby Duncan and M. J. Stillman 2254 FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS metal speciation after a few seconds of equilibration. The spectroscopic data were acquired in this study after a 2–5 min equilibration period at room tempera- ture, to ensure that thermodynamic equilibrium was achieved. The CD spectra measured during the metal- binding reaction (Fig. 2A) at pH 7.3 show a concomit- ant increase in CD signal intensity at 250 and 263 nm with the addition of up to 2.4 molar equivalents of Cd 2+ before a derivative-shaped signal, with band maximum at 263 nm, begins to dominate at 3.2 equiv- alents of Cd 2+ (Fig. 2A, inset). Finally, the full com- plement of 4.0 molar equivalents of Cd 2+ is required for the strong derivative signal to be observed with DA 220 reaching positive values. The UV absorption spectra (Fig. 2B) show an incremental increase in sig- nal intensity at 250 nm with the addition of Cd 2+ to the protein solution, reaching a maximum intensity at 4.0 molar equivalents of Cd 2+ , thus confirming the metal-binding ratio of Cd 4 (S cys ) 11 . Previous reports have shown that the intermediate Cd 1 a,Cd 2 a and Cd 3 a species each result in a mono- phasic CD spectrum with positive extrema at 250 nm, whereas the Cd 4 a species results in a derivative-shaped signal with positive and negative extrema at 260 nm and 240 nm, respectively, and a point of inflection at 250 nm, which was explained as being due to exciton splitting between the symmetric pairs of [Cd(S cys ) 4 ] 2 groups in the Cd 4 (S cys ) 11 binding site [27]. As noncoop- erative metal binding is predicted to result in the for- mation of intermediate, partially metallated, species, alaalaalaala lys gly met sergly A M 4 (S cys ) 11 Domain of Recombinant Human MT 1 2 7 6 5 4 11 8 3 10 9 3a 2a 4a 1a B C Fig. 1. (A) Sequence of the a domain of rhMT-1a, showing the connectivities of the four divalent metal cations to the 11 cystei- nyl sulfurs. The numbering of the cysteines (1–11 starting from the N-terminus) and the four divalent metals (1a)4a) are consistent with the metal–thiolate cluster shown in (C). (B) Space-filling and ribbon representations of the Cd 4 a-rhMT, emphasizing the left-han- ded wrapping of the polypeptide backbone around the metal–thiolate cluster. (C) Isola- ted Cd 4 (S cys ) 11 cluster present in the a domain of human MT-1a. The numbering of the cadmium and sulfur atoms corres- pond to those in the amino acid sequence shown in (A). Gray ¼ C; white ¼ H; blue ¼ N; red ¼ O; green ¼ Cd; yellow ¼ S. Diagram adapted from Chan et al. [44]. K. E. Rigby Duncan and M. J. Stillman Noncooperative metallation of metallothionein FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS 2255 these are qualitatively identifiable in the CD spectrum. As is clearly observed in Fig. 2A, addition of less than 4.0 molar equivalents of Cd 2+ results in CD spectra consistent with those observed for partially metallated domain species, supporting the model of a noncooper- ative metallation mechanism. Although a distinction between partially metallated intermediates and the fully metallated holoprotein can be made on the basis of the acquired CD spectra, quantitative analysis of the exact species being formed in the metallation reac- tion requires supplementary MS analysis. Figure 3 shows the corresponding MS data for the titration of apo-a-rhMT-1a with Cd 2+ at pH 7.8 fol- lowing a 2–5 min equilibration period at room tem- perature following each metal addition. The spectra on the left side of Fig. 3 are the original mass spectra, with mass ⁄ charge (m ⁄ z) values on the x-axis illustra- ting the charge state distributions of the protein spe- cies. The spectra on the right side of Fig. 3 are the deconvoluted spectra showing the mass and identity of the species detected. The deconvoluted spectra on the right side of Fig. 3 clearly show the formation of inter- mediate Cd 1 a,Cd 2 a and Cd 3 a species, with the Cd 4 a species forming only after > 3 equivalents of Cd 2+ have been titrated. Addition of 4.0 equivalents is required for 100% abundance of the Cd 4 a species (Fig. 3F), which correlates well with the sharp deriv- ative signal in the corresponding CD spectrum. At each molar equivalent addition of Cd 2+ , the ratio of the relative abundances of all cadmium-coordinated species to the total abundance of protein detected in the ESI mass spectrum correlated well with the total amount of Cd 2+ added, confirming that all of the Cd 2+ that was titrated into the solution was coordina- ting to the protein. Discussion In this report, we have unequivocally shown by CD spectroscopy and ESI MS that metal binding to the a domain of human MT-1a is a noncooperative process at physiologic pH. This implies that the four equilib- rium constants describing the sequential metallation reaction are decreasing in magnitude (K 1 > K 2 > K 3 > K 4 ), albeit only marginally, as the reaction does proceed to completion upon addition of 4.0 equiva- lents of Cd 2+ . The previously described metallation of the two-domain protein by Co 2+ indicated a simulta- neous metallation of the a and b domains, with two metal ions populating the a domain, and one in the b domain [25]. All three of these metal ions were shown to bind to independent tetrahedral tetrathiolate sites within the two domains. This was followed by coordi- nation of the fourth and fifth metal ions to the a domain for completion of the metallation of this domain prior to filling of the b domain. This work, as well as previous work on the metallation of MT with Cd 2+ [23], indicates that the mechanism may be sim- ilar to that of Co 2+ . The fact that the equilibrium con- stants for the a domain are greater than those for the b domain may be a factor in explaining the observed metal ion distribution. After coordination of the first two metal ions to the a domain in independent tetra- thiolate sites, the choice for the third incoming metal ion would be to form a bridging interaction in the a domain or to form another independent tetrathiolate site in the b domain. It is probable that the equilib- rium constant for the coordination of the first metal ion in the b domain (K 1b ) as an independent tetrathio- late site is greater than the equilibrium constant for the third metal ion in the a domain (K 3a ) with bridging A B Fig. 2. CD (A) and UV (B) absorption spectra of the titration of apo-a-rhMT-1a with Cd 2+ at pH 7.3. Spectral changes were recorded as up to 4.0 equivalents of Cd 2+ (3.3 mM) were titrated into a solution of apo-a-rhMT- 1a (15 l M)at22°C. Spectra were recorded at molar equivalent values of 0.0, 0.8, 1.6, 2.4, 3.2 and 4.0 of Cd 2+ at 22 °C. Inset: Plot of changes in CD intensity monitored at 223, 240, 250 and 263 nm as a function of molar equivalents of Cd 2+ up to a maximum of 4.0 equivalents. Noncooperative metallation of metallothionein K. E. Rigby Duncan and M. J. Stillman 2256 FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS ligands, especially as the noncooperative metal binding dictates that the K eq must be decreasing as the sequen- tial reaction proceeds. Finally, K 3a and K 4a for the a domain would have to be greater than K 2b and K 3b for the b domain, to explain the observed filling of the a domain prior to that of the b domain. Although the metal-binding reaction has been shown to proceed noncooperatively, this does not mean that a distinct order of metal binding to each of the four sites in the domain does not still exist. The fact that the polypeptide backbone adopts only one conforma- tion around the metal–thiolate cluster with specific connectivities does suggest that both the sequential metal-binding and metal-dependent protein-folding mechanisms occur in an energy-directed way. The fact that we have been able to detect the intermediate spe- cies in the metallation reaction indicates that the order of metal binding will one day be elucidated. In fact, strong evidence already exists for the site of the initial metallation reaction, a proposal first made by Robbins et al. upon elucidation of the crystal structure of rat liver MT-2 [15]. They stated that the most likely metal- lation site for the coordination of the first metal ion would be the four cysteine residues at the C-terminus of the protein, which are the only four consecutive cysteines to coordinate a single metal ion within the metal–thiolate cluster. This hypothesis was further sup- ported in a study by Munoz et al. [28] through investi- gation of a small peptide fragment corresponding to the C-terminal residues 49–61 of rabbit liver MT-2a, which encompassed the four consecutive cysteine resi- dues. The results showed the ability of the peptide to coordinate a single metal ion, which induced a metal- dependent fold of the peptide in the same configur- ation as the holoprotein. Finally, results from a computational molecular dynamics study carried out A B C D E F Fig. 3. ESI mass spectra of the titration of apo-a-rhMT-1a with Cd 2+ at pH 8.0. Spectral changes were recorded as aliquots of Cd 2+ (3.3 mM) were titrated into a solution of apo-a-rhMT-1a (21 l M)at22°C. Spectra were recorded at Cd 2+ molar equivalent val- ues of (A) 0.0, (B) 0.8, (C) 1.6, (D) 2.4, (E) 3.2, and (F) 4.0. The left side of the figure shows the measured mass spectra labeled with the charge states of the molecular spe- cies. The right side of the figure shows the deconvoluted spectra with the reconstruc- ted masses that correspond to the meas- ured spectra. Calculated mass: Cd 4 a-rhMT, 4083.0 Da; Cd 1 a-rhMT, 4193.4 Da; Cd 2 a-rhMT, 4303.8 Da; Cd 3 a-rhMT, 4414.2 Da; Cd 4 a-rhMT, 4524.6 Da. K. E. Rigby Duncan and M. J. Stillman Noncooperative metallation of metallothionein FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS 2257 on the a domain of human MT-1a [29] showed that the single remaining metal ion in the demetallation reaction was the C-terminal metal ion, indicating that occupancy of this metal site resulted in the least strain on the complex, and thus the lowest strain energy. The subsequent metal-binding order of the remaining three metal ions in the a domain still requires elucidation. The detection of stable, partially metallated domain intermediates in the sequential metallation pathway of MT is sufficient evidence to implicate a potential role for these species in vivo. Specifically, reconstitution of apo-Zn enzymes by Zn 7 -MT has been shown to occur readily in vitro, the most well-studied being apo-car- bonic anhydrase [30–33], and has been predicted to occur in vivo on the basis of analysis of the Zn 2+ pools in Erlich cells [34,35]; however, the fate of MT after metal ion donation has not been determined. Degrada- tion by cooperative demetallation of the remaining six metal ions following the loss of the first Zn 2+ would be, overall, an energetically expensive process, and would therefore be expected to be highly unfavorable. However, the demonstrated stability of the partially metallated species in this study provides support for the alternative scenario in which the partly demetall- ated MT product persists in vivo following metal ion donation. But if this is true, then what happens to the remaining metal ions that are bound to the MT mole- cule? Investigation into how the domain reacts in the event of metal ion donation will be of significant value for understanding the role of MT in the cellular meta- bolism of Zn 2+ . Despite the relatively large thermo- dynamic stability of the metal–thiolate clusters in MT, the metals have been shown to be kinetically labile in terms of both intramolecular and intermolecular metal exchange reactions [36]. Thus, it is probable that a spe- cific metal site is more labile than the others, and will therefore be the preferred site of demetallation. Kinetic studies of Zn 2+ extraction from Zn 7 ba-MT and Zn 4 a,Ag 6 b-MT demonstrated that the two domains differ with respect to the lability of the zinc ions and that, despite the increased thermodynamic stability of the a domain with Zn 2+ over the b domain, the Zn 2+ sites in the a domain were shown to be more labile [37]. It is possible that upon loss of the first metal ion, the three remaining metal ions in the a domain rear- range, either independently or in conjunction with the b domain, to position another metal ion into the more labile site in preparation for donation to another metal-requiring apo-enzyme. A considerable amount of effort has been directed in the recent past to understanding the mechanism of metal ion donation to apo-Zn 2+ -requiring enzymes, with the most detailed proposal involving a redox cycle in which oxidative release of Zn 2+ from Zn 7 -MT occurs by the formation of disulfide or S–O bonds upon interaction with cellular oxidants [38–40]. This proposal, however, is based on the assumption that the metallation mechanism of apo-MT is cooperative, and as such, only the metal-free and fully metallated holoprotein are present in vivo [41]. Although strong evidence exists for a critical balance between the MT ⁄ thionein pair [42,43] the evidence presented in this article demonstrates that alternative mechanisms for Zn 2+ probably exist. Moreover, the highly reducing environment of the cell, in which concentrations of reduced glutathione as high as 3 mm have been detec- ted, supports the theory generated by the data presen- ted, in which partially metallated, yet reduced, forms of MT can readily exist in the cell. In fact, the non- cooperative metallation and the subsequently decrea- sing equilibrium constants indicate that, from a coordination chemistry point of view, it is not only acceptable, but probable, that MT exists with a vacant site in vivo in the presence of limiting concentrations of free group 11 and 12 metal ions. Thus, it is proposed that MT only resides in the fully metallated holopro- tein upon influx of excess free metal ions into the cell. Upon overproduction of the metal-free protein in response to the influx, redistribution of the metal ions results in an average of less than the full complement of seven metals, a situation encountered in prepara- tions of rabbit liver MT, where excess metals are used for induction and subsequent isolation. The implica- tion of this proposal is that those metal ions that are sequestered by the protein could be holding the poly- peptide in a stable conformation, allowing the free thi- olate ligands to carry out vitally important chemistry in the cell. Specifically, MT has been implicated more recently in antioxidative, antiapoptotic and anti- inflammatory roles in vivo through reaction of the cysteine sulfur groups with reactive oxygen species, primarily in the brain and heart organs [7–11]. In summary, the metal-binding reactivity of the iso- lated a domain of human MT-1a was investigated, with specific emphasis on resolving the debate concern- ing the cooperative nature of the metal-binding mech- anism. The metallation reaction of the metal-free a domain with Cd 2+ was determined to proceed by a noncooperative mechanism by the detection of parti- ally metallated intermediate species under limiting Cd 2+ conditions. These species are predicted to be sta- ble in vivo and may even be the predominant form of MT in the cell, due to the very strict regulation of free metal ions. The vacant metal site(s) in the partially metallated species offer free cysteinyl thiolate ligands in the reducing environment of the cell for scavenging Noncooperative metallation of metallothionein K. E. Rigby Duncan and M. J. Stillman 2258 FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS of damaging reactive oxygen species, which supports the proposal of MT as a potent antioxidant and anti- apoptotic protein. Experimental procedures Materials The chemicals used were: cadmium sulfate (Fisher Scientific, Ottawa, ON, Canada); ultrapure Tris buffer (ICN Biomole- cules, Irvine, CA, USA); ammonium formate buffer (Ald- rich, Oakville, ON, Canada); isopropyl-b-d-thiogalactoside (Sigma-Aldrich, Oakville, ON, Canada); ammonium hydrox- ide (BDH Chemicals ⁄ VWR, Mississauga, ON, Canada); for- mic acid (J. T. Baker Chemical Co., Phillipsburg, NJ, USA); and hydrochloric acid (Caledon, Georgetown, ON, Canada). All solutions were made with >16 MWÆcm )1 deionized water (Barnstead Nanopure Infinity, Dubuque, IA, USA). HiTrap TM SP HP ion exchange columns (Amersham Bio- sciences ⁄ GE Healthcare, Piscataway, NJ, USA), superfine G-25 Sephadex (Pharmacia ⁄ Pfizer, Oakville, ON, Canada) and a stirred ultrafiltration cell (Amicon Bioseparations ⁄ Millipore, Bedford, MA, USA) with a YM-3 membrane (3000 MWCO) were used in the protein purification steps. Protein preparation The recombinant a domain of human MT-1a (sequence shown in Fig. 1A) was produced by overexpression in E. coli BL21(DE3) cells as an S-tag fusion protein. The cells were grown at 37 °C in LB medium containing 50 lm CdSO 4 and 50 lgÆmL )1 of kanamycin. Protein overexpres- sion was induced at an A 600 of 0.4–0.6 by the addition of isopropyl-b-d-thiogalactoside (final concentration 0.7 mm). The protein product was stabilized by the addition of CdSO 4 30 min after isopropyl-b-d-thiogalactoside induction to a final concentration of 200 lm. The cells were harvested by centrifugation at 7459 g for 15 min using an Avanti J-series centrifuge (Beckman Coulter, Mississauga, ON, Canada) with JLA-9.1000 rotor, resuspended in 10 mm Tris ⁄ HCl buffer (pH 7.4), and lysed with a French press. The lysed cellular fraction was centrifuged at 20 442 g for 40 min using an Avanti J-series centrifuge with JLA-25.50 rotor to remove the cellular debris. The supernatant was loaded onto an SP ion exchange cartridge for protein separ- ation, and the column was washed with argon-saturated 10 mm Tris ⁄ HCl buffer (pH 7.4). The Cd 2+ -substituted MT was eluted with a gradient of 5–20% NaCl in 10 mm Tris ⁄ HCl (pH 7.4). Protein fractions were collected on the basis of strong UV absorption at 250 nm corresponding to the ligand-to-metal charge transfer transitions of the SfiCd of the metal–thiolate clusters. The pooled protein fractions collected from the SP ion exchange column were concentra- ted to a volume of 15 mL using the Amicon ultrafiltration cell with a YM-3 cellulose membrane (3000 MWCO) under N 2 pressure. The S-tag was cleaved from the concentrated protein fraction using a Thrombin CleanCleave TM Kit (Sigma) by stirring the protein with the thrombin-coated beads under argon overnight at 4 °C. The cleaved protein was separated from the thrombin beads, and eluted from a superfine G-25 Sephadex column with Ar-saturated 10 mm Tris buffer (pH 7.4) to desalt prior to loading onto the SP ion exchange column for purification. The fractions collec- ted from the SP were pooled and concentrated to 8 mL, using the Amicon ultrafiltration cell. Further protein preparation for metal-binding studies Metal-free apo-a-rhMT was prepared by eluting the throm- bin-cleaved Cd-bound protein from a G-25 column equili- brated with a low-pH eluant. Apo-MT prepared for the CD studies was eluted with 10 mm Tris ⁄ HCl (pH 2.7), whereas the apo-MT prepared for the MS studies was elut- ed with deionized water adjusted with HCOOH to pH 2.8. Elution of the protein with a low-pH eluant effectively removes the metal ions from the protein; they separate from the protein band through the size-exclusion processes on the column. Preparation of apo-MT by this method sim- ultaneously desalts the solution by the same size-exclusion process. As MT is devoid of aromatic amino acids, the metal-free protein fractions were detected by UV absorption at 220 nm, which corresponds to the electronic transitions generated by the polypeptide backbone. The apo-a-rhMT used for the metal-binding studies was found to have con- centrations ranging from 10 to 20 lm, as determined by UV absorption at 220 nm (e 220 ¼ 40 000 LÆmol )1 Æcm )1 ) and atomic absorption spectroscopy following complete metalla- tion with Cd 2+ . Cadmium solutions were prepared in 10 mm Tris ⁄ HCl (pH 7.4) (for CD studies) or 25 mm ammonium formate (pH 7.4) (for MS studies) to a final concentration of 3.0–3.3 mm as determined by atomic absorption spectroscopy. The final samples were thoroughly evacuated and Ar-saturated to remove the bulk of the oxy- gen from the solutions, in order to prevent oxidation of the metal-free protein upon raising of the pH for the metalla- tion studies. Metallation of apo-a-rhMT with Cd 2+ at pH 7 CD ⁄ UV absorption spectroscopy The pH of apo-a-rhMT solution (13 lm) was raised from 2.7 to 7.3 by the addition of 10% NH 4 OH prior to the addition of Cd 2+ (3.3 mm). Cd 2+ was added in 0.8 molar equivalent increments up to 4.0 equivalents, with thorough mixing after each titration. CD and UV absorption spectra were recorded at each addition after a 2–5 min delay, in which the reaction could reach equilibrium conditions. K. E. Rigby Duncan and M. J. Stillman Noncooperative metallation of metallothionein FEBS Journal 274 (2007) 2253–2261 ª 2007 The Authors Journal compilation ª 2007 FEBS 2259 MS The pH of apo-a-rhMT solution (20 lm) was raised from 2.8 to 7.8 by the addition of 10% NH 4 OH prior to the addition of Cd 2+ (3.3 mm). Cd 2+ was added in 0.8 molar equivalent increments up to 4.0 equivalents, with thorough mixing after each titration. Mass spectra were acquired at each addition after a 2–5 min delay, in which the reaction could reach equilibrium conditions. Analytical and spectroscopic measurements CD spectroscopy CD spectra were acquired on a Jasco J810 spectropolarime- ter in a 1 cm quartz cuvette at room temperature (22 °C) and recorded using spectra manager v.1.52.01 (Jasco Inc., Easton, MD, USA) software. The wavelength range of 200–300 nm was scanned continuously at a rate of 50 nmÆmin )1 with a bandwidth of 2 nm. All spectra were baseline corrected with 10 mm Tris ⁄ HCl. The spectral data were organized and plotted using origin v.7.0383. UV absorption spectroscopy UV spectra were acquired on a Cary 5G UV-Vis-NIR spectrophotometer (Varian Canada Inc., Mississauga, ON, Canada) in a 1 cm quartz cuvette at room temperature (22 °C) and recorded using the cary win uv scan soft- ware application. The wavelength range of 200–300 nm was scanned continuously. All spectra were baseline cor- rected with 10 mm Tris ⁄ HCl. The spectral data were organized and plotted using origin v.7.0383. MS Mass spectra were acquired on an ESI-TOF mass spectro- meter (Waters Micromass Inc., Mississauga, ON, Canada) at room temperature (22 °C), and recorded using the mass lynx v.4.0 software package. The ESI-TOF mass spectro- meter was calibrated with a solution of NaI. The scan con- ditions for the spectrometer were: capillary, 3000.0 V; sample cone, 39.0 V; RF lens, 450.0 V; extraction cone, 11.0 V; desolvation temperature, 20.0 °C; source tempera- ture, 80.0 °C; cone gas flow, 51 LÆh )1 ; and desolvation gas flow, 528 LÆh )1 . The m ⁄ z range was 500.0–1600.0, the scan mode was continuum, and the interscan delay was 0.10 s. The observed spectra were reconstructed using the max ent 1 program from the mass lynx v.4.0 software package. Acknowledgements We thank NSERC of Canada for financial support (M. J. Stillman) and Postgraduate Scholarship (K. E. Rigby Duncan). We also thank Professor R. J. Pudde- phatt for use of the ESI mass spectrometer, funded by the Canada Research Chair program, and Doug Hair- sine for advice and discussion on the operation of the ESI mass spectrometer. References 1 Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181, 223–230. 2 Dill KA (1990) Dominant forces in protein folding. 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