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Insights into the thermodynamics of copper association with amyloid-b,

a-synuclein and prion proteinsw
Lian Hong* and John D. Simon*
Received 24th September 2010, Accepted 8th November 2010
DOI: 10.1039/c0mt00052c

This review examines recent studies on the thermodynamics of copper association with amyloid-b,
a-synuclein and prion protein, with an eye towards using this information to understand the
Published on 29 November 2010 on http://pubs.rsc.org | doi:10.1039/C0MT00052C

etiology of associated neurodegenerative diseases. A variety of binding affinities and binding sites,
which are essential to understand the function and consequence of copper-protein interaction,
have been reported for copper to these three neurobiologic systems. This current review reconciles
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the disparate models presented in the literature.

Introduction elevated copper concentrations have been observed in the rim

A characteristic feature of neurodegenerative diseases is the
formation of insoluble abnormal protein deposits in the
human brain, including the senile plaques of Alzheimer disease
(AD), the Lewy bodies of Parkinson’s disease (PD) and the
plaques of prion disease (PrPD). The protein contents of these
deposits have been determined, with the major protein
components being amyloid-b (Ab) peptide in the senile
plaques of AD,1,2 a-synuclein (aS) protein in the Lewy bodies
of PD,3 and prion protein (PrP) in the plaques of PrPD.4
Growing evidence suggests that the transition metal copper,
the 3rd most abundant transition metal ion in the brain, has a
substantial connection to the protein deposits and etiology of
these neurological diseases.5–11
The interactions of Cu2+ with Ab, aS and PrP have been
extensively studied in efforts to understand the mechanism of
the amyloid formation process and develop therapy for the
associated diseases. Ab, aS and PrP can all bind Cu2+;12–16
Department of Chemistry, Duke University, Durham, NC, 27708,
USA. E-mail: Lian.hong@duke.edu, john.simon@duke.edu
w This article is published as part of a themed issue on Metals in
Neurodegenerative Diseases, Guest Edited by David Brown.
of senile plaques of AD patients5 and the cerebrospinal fluid of
PD patients.6 In vitro studies show that Cu2+ can accelerate
the aggregation of these proteins/peptides.7–11 These interactions
have been reviewed from a variety of perspectives.10,11,17–21
Here we will focus on the thermodynamic properties,
specifically the binding affinity and binding enthalpy, for the
interaction of Cu2+ with Ab, aS and PrP and discuss the
connections between their thermodynamic properties and
coordination spheres.
Cu2+ in neutral buffer solution
Before discussing the interaction of Cu2+ with proteins, we
shall address one important issue that has failed to attract
adequate attention in the bioinorganic field: the hydrolysis of
Cu2+ in neutral buffer solution. The solubility product of
Cu(OH)2 (s) is B10 19–10 20 (NIST). Thus at neutral
pH B 7.4, copper in the form of aqueous Cu2+ has a
concentration of less than 10 mM. If the total copper concen-
tration in a buffer solution at pH 7.4 is higher than this level,
some of the copper ions hydrolyze and form copper hydro-
xides even though precipitation is not always observed.
The binding of a ligand to copper in such a solution cannot

Lian Hong received her BS John D. Simon received a BA

degree in Chemical Engineering from Williams College and
from Nanjing University, MS a PhD from Harvard
degree in Physical Chemistry University. After a post-
from Peking University, and doctoral fellowship with
PhD degree in Physical Professor M. A. El-Sayed at
Chemistry from Duke UCLA, he joined the faculty
University. She is a Research at UCSD. In 1998 he moved to
Scientist working in the Duke as the George B. Geller
Department of Chemistry at Professor of Chemistry. He
Duke University. Her emphasis served as department chair
in the lab is characterizing from 1999–2004 and is
metal–protein interactions. currently Vice Provost for
Academic Affairs. His group
Lian Hong John D. Simon studies the binding of reactive
metals to proteins and peptides
associated with neurodegenerative diseases, and structure and
function of human pigments.

262 Metallomics, 2011, 3, 262–266 This journal is c The Royal Society of Chemistry 2011

be taken simply as the binding of ligand to aqueous Cu2+; the
association of the OH ion to Cu2+ and the kinetics of
replacing OH by the ligand must be considered.
To demonstrate the importance of this association, we
examined the binding of EDTA to Cu2+ with and without
the presence of the weak Cu2+ ligand glycine, which stabilizes
Cu2+ in solution at pH 7.4. The binding of EDTA to Cu(Gly)2
in HEPES buffer at pH 7.4 gives a large binding constant (K)
(which is too strong to be determined accurately via the
isotherm), a 1 : 1 binding stoichiometry (N) and a binding
enthalpy (DH) of 6.0 kcal mol 1. These values are in excellent
agreement with literature values from NIST: N B 1 : 1 and
Published on 29 November 2010 on http://pubs.rsc.org | doi:10.1039/C0MT00052C
DH B 6.9 kcal mol 1. However, for the binding of EDTA
to Cu2+ without glycine, the binding parameters were determined
to be N B 0.5, K B 107 M 1 and DH B 14 kcal mol 1.
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These results are quite different from literature data for the
binding of EDTA to aqueous Cu2+ at pH 7.4 in PIPES buffer:
n B 1 : 1, K B 1016 M 1 and DH B 7.0 kcal mol 1. These
results clearly demonstrate the significant effects of Cu2+
hydrolysis on the thermodynamics of Cu2+–ligand interaction.
Thus care must be taken when studying the Cu2+–protein
interaction at near-physiological conditions (pH 7.4). Adding
a weak Cu2+ ligand to stabilize Cu2+ in solution is suggested
when a relatively high Cu2+ concentration is needed.
With the presence of a weak ligand, one also must take into
account the competition between the binding of Cu2+ to the
weak ligand and the biological system of interest. Many weak
ligands, e.g. glycine, histidine, and TRIS, associated with
Cu2+ in stoichiometries greater than 1 : 1 and so multiple
equilibria must be considered. All equilibria with dissociation
constants smaller than the free ligand concentration should be
considered. The optimal correction depends on the experimental
conditions. For experiments conducted with a constant free
ligand concentration, the correction of binding constant K
(the reciprocal of dissociation constant Kd) can be done by
multiplying the apparent constant by the binding polynomial
of the weak ligand: 1 + K1[L] + K1K2[L]2+. . ., where Ki is
the constant of the ith ligand binding to Cu2+ and [L] is the
free ligand concentration. If the free ligand concentration
changes during the experiment, numerical methods are
generally needed to correct for its competition. The general
rules used for developing correction methods utilize the charge
and mass balances of the materials in solution. For instance,
we used mass balance equations to correct for the competition
of glycine binding to Cu2+ in the isothermal calorimetry
titration of protein with Cu–Gly complex.12,22 In brief, the
mass balance equations of copper, protein, and glycine
were written out. The concentrations of each species were
determined by solving those equations, and the two steps of
glycine binding to Cu2+ were taken into account explicitly.
Cu2+ binding enthalpy and the number of protons exchanged
upon binding can provide insights into the Cu2+ coordination
sphere
In our recent work, we showed that the binding enthalpy (DH)
and number of protons exchanged between the peptide/
protein and the solvent (n) are dependent on the Cu2+
binding sites.13 Some common anchoring sites for Cu2+ in a
This journal is c The Royal Society of Chemistry 2011
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histidine-containing protein/peptide are the N-terminal amine
(NH2), amide CQO, amide N (N ), side chain COO
(Asp, Glu) and side chain imidazole N (NIm) of histidine. At
pH 7.4 and 37 1C, 60% of the N-terminal amines, 100% of the
amide N and 20% of the imidazole N are protonated while
CQO and COO have no ionizable protons.13 Coordination
of Cu2+ to moieties that exist in an equilibrium between
deprotonated and protonated states causes dissociation of
protons. A coordination sphere with a certain combination
of these sites therefore releases a certain number of protons.
For a coordination sphere without a complicated chelating
ring structure, the enthalpy can also be estimated. Binding
enthalpy can be experimentally determined by calorimetry,
where the ionization of the buffer also contributes. Because
most calorimetry studies we discuss in the following text were
conducted in PIPES buffer, we will use published data in
PIPES (buffer ionization enthalpy of 3.0 kcal mol 1, NIST)
for comparison. The enthalpy for Cu2+ binding to an ionized
N-terminal amine and a nearby COO or CQO can be
approximated as Cu2+ binding to one amino acid (except
for histidine), which has an enthalpy of B 6.0 kcal mol 1
(NIST). For the protonation of the N-terminal amine
(DHprotonation B 10 kcal mol 1, NIST) and buffer ionization,
the enthalpy for Cu2+ binding to the N-terminal amine and
COO /CQO in PIPES buffer is B 1.8 kcal mol 1 at pH 7.4.
Similarly the enthalpy for Cu2+ binding to the imidazole N
(His) and the amide N at pH 7.4 in PIPES buffer is suggested
to be around 5.8 kcal mol 1 and 3.0 kcal mol 1,
13
respectively. The enthalpy and number of protons released
from a particular coordination can be roughly estimated from
these values. For example, n and DH for a coordination of
{NH2, O, NIm, N } at pH 7.4 in PIPES are calculated to be
1.8 and B 4.6 kcal mol 1 respectively. This calculation is in
excellent agreement with the experimental data: a range of
n B 1.8–1.9 and DH B 1.7–3.8 kcal mol 1 has been observed
for five different coordinations with this structure in various
Ab mutants.13 Therefore, we suggest that although there might
be some error induced when there is stabilization of 5-,
6-member rings or bond constraints when multiple rings are
formed, the significant difference of binding enthalpies and
numbers of protons between these nitrogen sites make the
identification of the N sites feasible and accurate.
Cu2+ binding to Amyloid-b (Ab)
Quantifying the binding affinity of Cu2+ to human Ab is
crucial for the design of metal-chelation therapy for AD. A
wide range of binding affinities (105 M 1–109 M 1) for Cu2+
to human Ab (1–40) or its fragments(1–16, 1–28) have been
reported.12,13,22–28 We attribute the wide published range of
affinities to the different techniques, solution conditions and
methods of resolving binding constants in these studies.
Studies using Cu2+ solution in buffers without the presence
of a weak Cu2+ ligand gave binding constants B 105 M 1 at
pH 7.2–7.4.23,24 This number is likely much smaller than the
real binding constant at this pH, due to the hydrolysis of
Cu2+. Studies using competing ligands or in buffers with weak
Cu2+ affinity (e.g. glycine and TRIS) also have reported a
wide range of affinities: 105 M 1–109 M 1, likely reflecting the
Metallomics, 2011, 3, 262–266 263

different or absent corrections for the competition between
the peptide and the coordinated weak ligands. Through
ESI-MS studies, we did not reveal the presence of Ab-Cu–Gly
complexes.12 We developed thermodynamic models to rigorously
take into account the glycine competition in determining the
Cu2+-Ab binding affinity from the isothermal calorimetry
titration of Ab with the Cu2+–glycine complex,12,13,22 obtaining
an affinity of Cu2+ to human Ab(16) of 3.1  109 M 1 at
pH 7.4 and 37 1C. Using the same methodology, we reconciled the
constants determined for Cu2+ to human Ab in the presence
of glycine in two other studies.25,26 There remain some
discrepancies for the data in TRIS buffer.27,28 In a recent
Published on 29 November 2010 on http://pubs.rsc.org | doi:10.1039/C0MT00052C
review, Faller et al. suggested that the binding of TRIS to
Cu2+ is not fully understood, which prevents accurately
modeling the binding of the metal to the buffer. It is therefore
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suggested that the data obtained from experiments using
glycine as a competing ligand are by far the most accurate
for Cu2+ addition to human Ab (109 M 1 at pH 7.4 and
1010 M 1 at pH 8.0).17 With the same method, we determined
the Cu2+-rat Ab affinity to be B1010 M 1 at pH7.4, B3 times
stronger than that for human Ab,13 the same trend as observed
by Kowalik-Jankowska et al.29
Structural information on Cu2+ coordination is essential to
understanding the Cu2+-induced aggregation of Ab. It is
generally accepted that the binding site at physiological
pH (B7.4) is predominantly a 3N1O structure according to
spectroscopic studies.26,29–34 However, there is no consensus
on the exact coordination spheres for Cu2+ bound to Ab. The
finding of two Cu2+ structures with distinct sets of EPR
parameters indicates the presence of two types of coordination
at equilibrium.26,34–37 Several studies assuming a major
coordination may present an average result from all the
coordinations30,38,39 and are not discussed in detail herein in
favor of focusing on studies containing efforts to identify each
coordination contributing to the equilibrium.
Kowalik-Jankowska et al. suggested that at pH 7.4,
complexes of Cu2+ bound to human Ab(1–16 or 1–28) are
almost equally distributed between two types of binding
geometries at physiological pH (7.4), {NH2, O, NImHis13,
NImHis14} and {NH2, O, NImHis6, N }.34 However, no specific
evidence was provided to support the identification of the
histidines for the former coordination. Viles’ group proposed
two coordination spheres both involving all three histidines
(His6, His13, and His14), two O-ligands and an N-terminus,
with the N-terminal amino group as an axial ligand at pH 7.4
but in the plane at pH 9.0.35 Two recent studies by the
Barnham group examined the coordination spheres with
CW-EPR and HYSCORE,36,37 and found that the two
components are present at pH 8.0 in comparable proportions.
Component I, prevalent at lower pH, is suggested to reflect
two coordination spheres with similar EPR properties,
as {NH2, COO , NImHis6, NImHis13} (Component Ia),
and {NH2, COO , NImHis6, NImHis14} (Component Ib).
Component II is assigned to the coordination {CO, NImHis6,
NImHis13, NImHis14}. However, it is worth noting that the
assignments by the Viles group and the Barnham group
cannot explain the pH dependence of the two structures that
was observed in the EPR spectra. As for rat Ab peptides,
the work of Kowalik-Jankowska et al. showed that at
264 Metallomics, 2011, 3, 262–266
View Article Online
near-physiological pH (7.4), the coordination of Cu2+ by rat
Ab(16) is {NH2, COO , NIm, N }.34 However, in a later work,
Gaggelli et al. suggested that both His6 and His14 of rat
Ab(28) serve as anchoring sites for Cu2+.40
In a recent work, we studied the coordination sphere of
Cu2+ in human and rat Ab with the aid of EPR and CD by
analyzing the binding enthalpies and numbers of protons
exchanged for Cu2+ binding to Ab and a library of Ab
mutants.13 In human Ab, three coordination spheres were
revealed at pH 7.4: {NH2, O, NImHis6, N }(50%), {NH2, O,
NImHis6, NImHis13} (30%) and {NH2, O, NImHis6, NImHis14}
(20%). In rat Ab at pH 7.4, however, one dominant
coordination {NH2, O, NImHis6, N } (>94%) is present.
The K, DH and n for Cu2+ binding to human Ab is estimated
to be 3.1  109 M 1, 6.1 kcal mol 1 and 1.5, respectively,
based on the three coordinations, which is in excellent
agreement with the experimental data (K = 3.0  109 M 1,
DH = 5.6 kcal mol 1 and n = 1.5).13 The pH-dependence
further confirmed our assignments of the coordinations in
human and rat Ab and their mutants.13 The single amino acid
difference, R5G, between human and rat Ab stabilizes the
coordination {NH2, O, NImHis6, N } in rat Ab. The formation
of {NH2, O, NImHis6, N } requires less conformational
rearrangement than that of {NH2, O, NImHis6, NImHis13} or
{NH2, O, NImHis6, NImHis14}. Therefore, the shift of the
equilibrium between coordinations caused by R5G substitution
is suggested to account for the decreased Cu2+-induced
aggregation in rat Ab. This speculation is confirmed by the
fact that the Cu2+-induced aggregation of R5G(40) is about
the same as that of rat Ab(40) but less than that of human
Ab(40).13
Cu2+ binding to a-synuclein protein (aS)
aS has the capacity to take up two Cu2+ ions, one strongly
bound at the N-terminus and one relatively weakly bound at
the COO -rich C-terminus.9 However, in the presence of a
pool of various proteins and amino acids in vivo, the weak
binding has little chance to occur. We therefore will focus on
the strong binding at the N-terminus. We examined the
binding thermodynamics of Cu2+ to WT aS and its two
mutants, A53T and A30P, by the isothermal calorimetry
titration of aS with Cu–Gly.14 ESI-MS studies did not reveal
any as-Cu–Gly complexes.14 In the presence of excess glycine,
which prevents the weak binding, we observed a 1 : 1 binding
ratio. The binding constants for Cu2+ to the three aS proteins
are similar, in the range of 3.6–5.0  109 M 1 at pH 7.4 and
37 1C. The slightly lower affinity of A30P (3.6  109 M 1 vs.
5.0  109 M 1) is suggested to be due to different confor-
mational arrangements for the binding of copper from those
of WT and A53T.
These binding constants are consistent with the work by
Wilkinson et al., in which an excess of glycine is added to
stabilize Cu2+,41 but is higher than those determined with
Cu2+ suspended in neutral buffer only (104 M 1 at pH 7.4 to
107 M 1).8,42,43 We suggest that the weak Cu2+ binding
to the C-terminus and the hydrolysis of Cu2+ contribute
to the smaller constant in these studies. The binding
enthalpies for Cu2+ to WT aS, A53T and A30P are similar
This journal is c The Royal Society of Chemistry 2011

( 1.7 to 1.8 kcal mol 1 at pH 7.4), implying that the direct
coordination spheres for the three aS proteins are the same. It
has been shown that both A30P and A53T mutations increase
the rate of aS oligomerization, but the rate of mature fibril
formation is increased by A53T and decreased by A30P.44 The
similarity of the thermodynamics suggests that, unlike the
situation in Ab, it is more likely that the mutation itself rather
than the copper coordination of the three aS proteins affects
the Cu2+-induced aggregation process of aS.
The coordination of Cu2+-WT aS was proposed to
be 2N2O and/or 3N1O at pH 6.5 7.4 according to EPR
characterization.9,45 It was debated whether the His50 and
Published on 29 November 2010 on http://pubs.rsc.org | doi:10.1039/C0MT00052C
the N-terminal cooperate to coordinate Cu2+.9,45,46 Rasia
et al. suggested a specific 2N2O coordination including the
cooperation of the N-terminal and His50 at pH 6.5, since
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DEPC modification of aS eliminated the binding to both
His50 and the N-terminus.9 A later work by Sung attempted
to challenge this cooperation based on the fact that
the mutation H50A failed to abolish Cu2+ binding to the
N-terminal.46 However, this result can only prove that the
N-terminal can bind to Cu2+ without the presence of His50; it
cannot eliminate the possibility of cooperation between the
N-terminal and His50.
Drew et al. proposed an equilibrium between two
coordinations, {NH2, COO , N } (45%) and {NH2, COO ,
N , NIm} (55%), for the Cu2+-aS complex at pH 7.4.45 We
have shown that the binding enthalpy and number of protons
can provide insights into the coordination. Unfortunately, the
number of protons released from aS upon Cu2+ association
was not determined. But the fact that the constant at pH 7.4 is
2 (>100.2, but o(100.2)2) times that at pH7.2 indicates that the
number of protons released is greater than 1 but smaller than
2. With a 2N2O/3N1O structure and 2 > n > 1, we conclude
that one of the N atoms must be the amide N, consistent with
the conclusion by Drew et al. The binding enthalpies of
{NH2, COO , N } and {NH2, COO , N , NIm} are estimated
to be B1.2 kcal mol 1 and 4.6 kcal/mole. Accounting for the
population of the two coordinations, the average enthalpy
must be B 2.0 kcal mol 1, which is consistent with the
measured enthalpy ( 1.8 kcal mol 1 for Cu2+ to WT aS in
PIPES, pH 7.4) A more accurate number of protons released
from aS and thermodynamic studies of H50A might provide
more definitive information on the coordination(s).
2+
Cu binding to prion protein
The N-terminal of PrP protein, PrP (60–91), has a
highly conserved region, with a four-octarepeat sequence,
(PHGGGWGQ)4.15 It was shown that each of the octapeptides
can bind one Cu2+ via the imidazole N and two nearby amide
N’s. Millhauser and coworkers showed that the binding of
Cu2+ to Ac-HGGGW has an EPR spectrum similar to that of
Table 1 The binding constants and proposed coordinations for Cu2+ to human Ab, aS and Prp proteins at pH 7.4
Human Ab
1 9 12,13,22
Binding constant/M 3  10
Proposed coordinations NH2, COO , NIm6, N },
{NH2, O, NIm6, NIm13},
{NH2, O, NIm6, NIm14}13
This journal is c The Royal Society of Chemistry 2011
View Article Online
Cu2+ bound to the single octapeptide and suggested it as a
model system for that binding.15,47 The affinity of Cu2+ to
such coordination is B 105–106 M 1. When the Cu : aS ratio
o1, or at acidic pH or in the presence of excess competing
ligand at neutral pH, Cu2+ binds to multiple His (>3), which
is of greater affinity than that for Cu2+ to a single imidazole
N.15,47,48 The affinity of Cu2+ to multihistidine (3–4 imidazole
N’s) coordination was determined by EPR to be 1010 M 1 via
the titration of Prp (23–28, 57–91) with Cu2+ in the presence
of excess amino acid ligands.15
There are a few studies suggesting a higher affinity for Cu2+
to PrP for the multihistidine coordination.16,49,50 Jackson et al.
reported the binding constant of Cu2+ to PrP(52–98) as
1014 M 1 in the presence of excess glycine.16 However, the
glycine dissociation constant was not corrected for the pH;
when corrected, the constant is 8  1010 M 1 at pH 8.0, which
is consistent with Millhauser’s work. Thompsett et al.
determined the constant by titrating Prp(23–231) using a
Cu–Gly complex in water and suggested a binding constant
of 1012 M 1 at pH 7.49 But this constant was corrected by
multiplying the apparent constant by KCu–Gly, when multiplying
by (1 + KCu–Gly[Gly] + KCu–Gly[Gly] KCuGly–Gly[Gly]) would
have been more appropriate.
A recent study examined the affinity of Cu2+ for mPrp via
the competition of glycine with Prp to bind with Cu2+, 51
finding an affinity of B 4  107 M 1. Unlike the use of excess
glycine by Millhauser et al., and Jackson et al., this work
varied the glycine concentration from 0–32 mole equivalences.
At low glycine concentration, the binding of Cu2+ to single
histidine and two near amide N’s is possible, and a more
complicated process must be considered. In this context,
we suggest that the affinity of Cu2+ to the multihistidine
coordination is more likely B 1010 M 1. In preliminary efforts
in collaboration with Millhauser, we found that the binding
enthalpy of Cu2+ to Prp (23–28, 57–91) is Br 15 kcal mol 1
in PIPES buffer at pH 7.4. This number suggests the contribution
of at least 3 imidazole N’s to the Cu2+ coordination, which is
consistent with previous EPR studies.15,47 However, due to
lack of a proper sequential model to fit the data, more accurate
values for the binding constant and binding enthalpy cannot
be extracted.
Conclusion
This review has focused on the thermodynamics (binding
constant, binding enthalpy) of Cu2+ binding to three
neurologically related proteins: Ab, aS and PrP. For each of
the three proteins, a wide range of binding constants has been
reported. We suggest that different techniques, different
solution conditions, and different ways to deconvolute
binding constants account for the large ranges of affinities.
aS PrP
9 14
(4–5)  10 1010 15
{NH2, COO , N }, {X, NIm, NIm, NIm}15,47
{NH2, COO , NIm50, N }45
Metallomics, 2011, 3, 262–266 265

To determine the affinity, we suggest adding a competing
ligand (e.g. glycine) to avoid the poorly-understood
Cu2+–hydroxide system. With appropriate corrections, we
have now reconciled the binding constants produced in the
presence of glycine. The binding constants and proposed
coordination spheres are summarized in Table 1. It is
intriguing that the three proteins/peptide have comparable
Cu2+ affinities, with binding constants in the range of 109
M 1–1010 M 1 at pH 7.4, which may imply a role for the
homeostasis in Cu2+ in the neurological diseases. The binding
enthalpy and the number of protons can be correlated to the
coordination sphere in histidine-containing proteins. A
Published on 29 November 2010 on http://pubs.rsc.org | doi:10.1039/C0MT00052C
comprehensive examination of the binding enthalpy of Cu2+
and the number of protons released for various coordinations
may provide a two-dimensional ruler for identification of
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coordination.
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