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].. 2A, addit
Trang 1a 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 [M4(Scys)11] and [M3(Scys)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 Cd2+ 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 Cd2+ con-ditions Titration with four molar equivalents of Cd2+was required for the formation of the Cd4a 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.
Trang 2view 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 M2+
(M¼ Zn, Cd) initially to the a domain, thereby
lead-ing to formation of the M4a cluster prior to formation
of the M3b 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 As3+ binding to the
two isolated domains were also interpreted in terms of
a series of noncooperative bimolecular reactions [24]
The fact that Cd2+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
Co2+[25] and Cd2+[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 Cd2+ (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 Cd4a-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 Cd4(Scys)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 Cd2+to apo-a-rhMT , the metal-free pro-tein will be detected together with a corresponding fraction of the metal-saturated Cd4a species if the met-allation mechanism proceeds by a positively cooper-ative pathway Alterncooper-atively, the partially filled Cd1a,
Cd2a and Cd3a 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 Cd2+ 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 Cd2+ 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 Cd2+can be considered a nearly instantaneous reaction In addition, no evidence has been reported to show that any change occurs to the
Trang 3metal 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
Cd2+ before a derivative-shaped signal, with band
maximum at 263 nm, begins to dominate at 3.2
equiv-alents of Cd2+ (Fig 2A, inset) Finally, the full
com-plement of 4.0 molar equivalents of Cd2+ is required
for the strong derivative signal to be observed with
DA220 reaching positive values The UV absorption
spectra (Fig 2B) show an incremental increase in
sig-nal intensity at 250 nm with the addition of Cd2+ to the protein solution, reaching a maximum intensity at 4.0 molar equivalents of Cd2+, thus confirming the metal-binding ratio of Cd4(Scys)11
Previous reports have shown that the intermediate
Cd1a, Cd2a and Cd3a species each result in a mono-phasic CD spectrum with positive extrema at 250 nm, whereas the Cd4a 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(Scys)4]2 groups in the Cd4(Scys)11binding site [27] As noncoop-erative metal binding is predicted to result in the for-mation of intermediate, partially metallated, species,
ala ala ala ala lys gly met ser gly
A M4(Scys)11 Domain of Recombinant Human MT
1
2 7 6
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 Cd4a-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].
Trang 4these are qualitatively identifiable in the CD spectrum.
As is clearly observed in Fig 2A, addition of less than
4.0 molar equivalents of Cd2+ 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 Cd2+ 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 Cd1a, Cd2a and Cd3a species, with the Cd4a
species forming only after > 3 equivalents of Cd2+
have been titrated Addition of 4.0 equivalents is
required for 100% abundance of the Cd4a species
(Fig 3F), which correlates well with the sharp
deriv-ative signal in the corresponding CD spectrum At
each molar equivalent addition of Cd2+, 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 Cd2+ added, confirming that all of the
Cd2+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 (K1> K2>
K3> K4), albeit only marginally, as the reaction does proceed to completion upon addition of 4.0 equiva-lents of Cd2+ The previously described metallation of the two-domain protein by Co2+ 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
Cd2+ [23], indicates that the mechanism may be sim-ilar to that of Co2+ 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 (K1b) as an independent tetrathio-late site is greater than the equilibrium constant for the third metal ion in the a domain (K3a) with bridging
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 m M ) were titrated into a solution of apo-a-rhMT-1a (15 l M ) at 22 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.
Trang 5ligands, especially as the noncooperative metal binding
dictates that the Keqmust be decreasing as the
sequen-tial reaction proceeds Finally, K3a and K4a for the a
domain would have to be greater than K2band K3bfor
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 Cd2+at pH 8.0 Spectral
changes were recorded as aliquots of Cd 2+
(3.3 m M ) were titrated into a solution of
apo-a-rhMT-1a (21 l M ) at 22 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: Cd4a-rhMT,
4083.0 Da; Cd 1 a-rhMT, 4193.4 Da;
Cd2a-rhMT, 4303.8 Da; Cd 3 a-rhMT,
4414.2 Da; Cd4a-rhMT, 4524.6 Da.
Trang 6on 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 Zn7-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 Zn2+pools
in Erlich cells [34,35]; however, the fate of MT after
metal ion donation has not been determined
Degrada-tion by cooperative demetallaDegrada-tion of the remaining six
metal ions following the loss of the first Zn2+ 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 Zn2+ 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 Zn2+ extraction from Zn7ba-MT and
Zn4a,Ag6b-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 Zn2+over the b domain, the Zn2+
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-Zn2+-requiring enzymes,
with the most detailed proposal involving a redox cycle
in which oxidative release of Zn2+ from Zn7-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
Zn2+ 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 metallapresen-ted, 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-bindconcern-ing mech-anism The metallation reaction of the metal-free a domain with Cd2+ was determined to proceed by a noncooperative mechanism by the detection of parti-ally metallated intermediate species under limiting
Cd2+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
Trang 7of 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 Chefor-mical Co., Phillipsburg, NJ, USA);
and hydrochloric acid (Caledon, Georgetown, ON, Canada)
All solutions were made with >16 MWÆcm)1deionized water
(Barnstead Nanopure Infinity, Dubuque, IA, USA)
HiTrapTM 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 37C in LB medium containing 50 lm
CdSO4and 50 lgÆmL)1of kanamycin Protein
overexpres-sion was induced at an A600 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
CdSO430 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 Cd2+
-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
N2 pressure The S-tag was cleaved from the concentrated protein fraction using a Thrombin CleanCleaveTM Kit (Sigma) by stirring the protein with the thrombin-coated beads under argon overnight at 4C 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 concentracollec-ted 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 deionizelut-ed water adjustelut-ed 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 (e220¼ 40 000 LÆmol)1Æcm)1) and atomic absorption spectroscopy following complete metalla-tion with Cd2+ 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 Cd2+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% NH4OH prior to the addition of Cd2+(3.3 mm) Cd2+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
Trang 8The pH of apo-a-rhMT solution (20 lm) was raised from
2.8 to 7.8 by the addition of 10% NH4OH prior to the
addition of Cd2+ (3.3 mm) Cd2+ 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 (22C)
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
(22C) 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 (22C), and recorded using the mass
lynxv.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, 20.0C; source
tempera-ture, 80.0C; 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
ent1 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 Biochemistry 29, 7133–7155
3 Yon JM (2001) Protein folding: a perspective for bio-logy, medicine and biotechnology Braz J Med Biol Res
34, 419–435
4 Creighton TE (1990) Protein folding Biochem J 270, 1–16
5 Bren KL, Pecoraro VL & Gray HB (2004) Metallo-protein folding Inorg Chem 43, 7894–7896
6 Stillman MJ, Shaw CF III & Suzuki KT (1992) Meta-llothioneins: Synthesis, Structure and Properties of Metallothioneins, Phytochelatins and Metal–Thiolate Complexes VCH Publishers, Inc., New York, NY
7 Aschner M (1996) The functional significance of brain metallothioneins FASEB J 10, 1129–1136
8 Hidalgo J (2004) Metallothioneins and brain injury: what transgenic mice tell us Environ Health Prev Med
9, 87–94
9 Hidalgo J, Aschner M, Zatta P & Vasak M (2001) Roles of the metallothionein family of proteins in the central nervous system Brain Res Bull 55, 133–145
10 Kang YJ (1999) The antioxidant function of meta-llothionein in the heart Proc Soc Exp Biol Med 222, 263–273
11 Wiese L, Kurtzhals JAL & Penkowa M (2006) Neuro-nal apoptosis, metallothionein expression and proin-flammatory responses during cerebral malaria in mice Exp Neurol 200, 216–226
12 Notta F & Koropatnick JD (2006) Metallothioneins in drug resistance In Cancer Drug Resistance (Teicher BA, ed.), pp 223–239 Humana Press, Totowa, NJ
13 Otvos JD & Armitage IM (1980) Structure of the metal clusters in rabbit liver metallothionein Proc Natl Acad Sci USA 77, 7094–7098
14 Otvos JD & Armitage IM (1979)113Cd NMR of meta-llothionein: direct evidence for the existence of poly-nuclear metal binding sites J Am Chem Soc 101, 7734–7736
15 Robbins AH, McRee DE, Williamson M, Collett SA, Xuong NH, Furey WF, Wang BC & Stout CD (1991) Refined crystal structure of cadmium–zinc
metallothionein at 2.0 A˚ resolution J Mol Biol 221, 1269–1293
16 Stillman MJ & Zelazowski AJ (1989) Domain-specificity
of Cd2+and Zn2+binding to rabbit liver
Trang 9metallothio-nein 2 Metal ion mobility in the formation of
Cd4-metallothionein a-fragment Biochem J 262,
181–188
17 Stillman MJ (1995) Metallothioneins Coord Chem Rev
144, 461–511
18 Winge DR & Nielson KB (1984) Formation of the
metal–thiolate clusters of rat liver metallothionein
Envi-ron Health Persp 54, 129–133
19 Nielson KB & Winge DR (1983) Order of metal binding
in metallothionein J Biol Chem 258, 13063–13069
20 Gehrig PM, You C, Dallinger R, Gruber C, Brouwer
M, Kagi JHR & Hunziker PE (2000) Electrospray
ioni-zation mass spectrometry of zinc, cadmium, and copper
metallothioneins: evidence for metal-binding
cooperativ-ity Prot Sci 9, 395–402
21 Good M, Hollenstein R, Sadler PJ & Vasak M (1988)
113
Cd NMR studies on metal–thiolate cluster formation
in rabbit Cd(II)-metallothionein: evidence for a pH
dependence Biochemistry 27, 7163–7166
22 Zaia J, Fabris D, Wei D, Karpel RL & Fenselau C
(1998) Monitoring metal ion flux in reactions of
meta-llothionein and drug-modified metameta-llothionein by
elec-trospray mass spectrometry Prot Sci 7, 2398–2404
23 Willner H, Vasak M & Kagi JHR (1987) Cadmium–
thiolate clusters in metallothionein: spectrophotometric
and spectropolarimetric features Biochemistry 26,
6287–6292
24 Ngu TT & Stillman MJ (2006) Arsenic binding to
human metallothionein J Am Chem Soc 128, 12473–
12483
25 Bertini I, Luchinat C, Messori L & Vasak M (1989)
Proton NMR studies of the cobalt(II)-metallothionein
system J Am Chem Soc 111, 7296–7300
26 Ejnik J, Robinson J, Zhu J, Forsterling H, Shaw CF III
& Petering DH (2002) Folding pathway of
apo-metal-lothionein induced by Zn2+, Cd2+and Co2+ J Inorg
Biochem 88, 144–152
27 Stillman MJ, Cai W & Zelazowski AJ (1987) Cadmium
binding to metallothioneins Domain specificity in
reac-tions of a and b fragments, apometallothionein, and zinc
metallothionein with Cd2+ J Biol Chem 262, 4538–4548
28 Munoz A, Laib F, Petering DH & Shaw CF III (1999)
Characterization of the cadmium complex of peptide
49–61: a putative nucleation center for
cadmium-induced folding in rabbit liver metallothionein IIA
J Biol Inorg Chem 4, 495–507
29 Rigby KE, Chan J, Mackie J & Stillman MJ (2006)
Molecular dynamics study on the folding and
meta-llation of the individual domains of metallothionein
Proteins 62, 159–172
30 Li T-Y, Kraker AJ, Shaw CF III & Petering DH (1980)
Ligand substitution reactions of metallothioneins with
EDTA and apo-carbonic anhydrase Proc Natl Acad Sci USA 77, 6334–6338
31 Udom AO & Brady FO (1980) Reactivation in vitro of zinc-requiring apo-enzymes by rat liver zinc-thionein Biochem J 187, 329–335
32 Huang Z-X, Liu F, Zheng Q & Wen W-H (2001) Zinc transfer kinetics of metallothioneins and their fragments with apo-carbonic anhydrase Chin J Chem 19, 462– 467
33 Ejnik J, Munoz A, Gan T, Shaw CF III & Petering DH (1999) Interprotein metal ion exchange between cad-mium-carbonic anhydrase and apo- or zinc-metallothio-nein J Biol Inorg Chem 4, 784–790
34 Kraker AJ, Krakower G, Shaw CF III, Petering DH & Garvey JS (1988) Zinc metabolism in Ehrlich cells: properties of a metallothionein-like zinc-binding protein Cancer Res 48, 3381–3388
35 Krezoski SK, Villalobos J, Shaw CF III & Petering DH (1988) Kinetic lability of zinc bound to metallothionein
in Ehrlich cells Biochem J 255, 483–491
36 Otvos JD, Lui X, Li H, Shen G & Basti M (1993) Dynamic aspects of metallothionein structure In Meta-llothionein III(Suzuki KT, Imura N & Kimura M, eds),
pp 57–74 Birkhauser-Verlag, Basel, Switzerland
37 Li H & Otvos JD (1998) Biphasic kinetics of Zn2+ removal from Zn metallothionein by nitrilotriacetate are associated with differential reactivity of the two metal clusters J Inorg Biochem 70, 187–194
38 Jiang L-J, Maret W & Vallee BL (1998) The glutathione redox couple modulates zinc transfer from metallothio-nein to zinc-depleted sorbitol dehydrogenase Proc Natl Acad Sci USA 95, 3483–3488
39 Maret W (1994) Oxidative metal release from metallo-thionein via zinc-thiol⁄ disulfide interchange Proc Natl Acad Sci USA 91, 237–241
40 Maret W & Vallee BL (1998) Thiolate ligands in meta-llothionein confer redox activity on zinc clusters Proc Natl Acad Sci USA 95, 3478–3482
41 Maret W (2004) Zinc and sulfur: a critical biological partnership Biochemistry 43, 3301–3309
42 Maret W (2003) Cellular zinc and redox states converge
in the metallothionein⁄ thionein pair J Nutr 133, 1460S– 1462S
43 Yang Y, Maret W & Vallee BL (2001) Differential fluorescence labelling of cysteinyl clusters uncovers high tissue levels of thionein Proc Natl Acad Sci USA 98, 5556–5559
44 Chan J, Huang Z, Merrifield ME, Salgado MT & Still-man MJ (2002) Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniques Coord Chem Rev
233, 319–339