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:
Website: />(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.
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