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The Palladium(II) Complex of Aβ 4−16 as Suitable Model for Structural Studies of Biorelevant Copper(II) Complexes of N-Truncated Beta-Amyloids

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The Aβ 4−42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aβ 4−16 and Aβ 4−9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aβ 4−x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aβ 4−16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aβ 4−16 , controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1 H-13 C HSQC and 1 H-15 N HSQC NMR experiments, for natural abundance 13 C and 15 N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aβ 4−16 , Pd(II)-Aβ 4−16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aβ complexes.
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International Journal of
Molecular Sciences
Article
The Palladium(II) Complex of Aβ416 as Suitable
Model for Structural Studies of Biorelevant
Copper(II) Complexes of N-Truncated Beta-Amyloids
Mariusz Mital 1, Kosma Szutkowski 2, Karolina Bossak-Ahmad 1, Piotr Skrobecki 1,
Simon C. Drew 1, Jarosław Pozna ´nski 1, Igor Zhukov 1,* , Tomasz Fr ˛aczyk 1,*
and Wojciech Bal 1,*
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warszawa, Poland;
1988.mariusz.m@gmail.com (M.M.); karolina.bossak@gmail.com (K.B.-A.); skrobec@gmail.com (P.S.);
scdrew1@gmail.com (S.C.D.); jarek@ibb.waw.pl (J.P.)
2NanoBioMedical Centre, Adam Mickiewicz University, 61-614 Pozna´n, Poland;
kosma.szutkowski@outlook.com
*Correspondence: igor@ibb.waw.pl (I.Z.); tfraczyk@ibb.waw.pl (T.F.); wbal@ibb.waw.pl (W.B.);
Tel.: +48-22-592-2038 (I.Z.); +48-22-592-2371 (W.B.)
Received: 12 November 2020; Accepted: 30 November 2020; Published: 2 December 2020


Abstract:
The A
β442
peptide is a major beta-amyloid species in the human brain, forming toxic
aggregates related to Alzheimer’s Disease. It also strongly chelates Cu(II) at the N-terminal
Phe-Arg-His ATCUN motif, as demonstrated in A
β416
and A
β49
model peptides. The resulting
complex resists ROS generation and exchange processes and may help protect synapses from
copper-related oxidative damage. Structural characterization of Cu(II)A
β4x
complexes by NMR
would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead
we used an isostructural diamagnetic Pd(II)-A
β416
complex as a model. To avoid a kinetic trapping
of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic
procedure for ATCUN Pd(II)A
β416
, controlled by CD, fluorescence and ESI-MS. Its assignments
and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY,
1
H-
13
C HSQC and
1
H-
15
N HSQC
NMR experiments, for natural abundance
13
C and
15
N isotopes, aided by corresponding experiments
for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest
of the peptide mostly unstructured. The diffusion rates of A
β416
, Pd(II)-A
β416
and their mixture
determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular
assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of
structural aspects of Cu(II)-Aβcomplexes.
Keywords:
Alzheimer’s disease; A
β
peptide; NMR spectroscopy;
13
C relaxation; Palladium(II);
ATCUN motif
1. Introduction
The A
β442
peptide is a major beta-amyloid species in the human brain. It was first co-discovered
in 1985 as a major component of amyloid plaques of Alzheimer’s Disease (AD) brains, and was found
to be more abundant than the commonly studied A
β142
and A
β140
peptides [
1
,
2
]. However, this
discovery was soon disregarded, and the research was focused on the latter two peptides as forming
neurotoxic aggregates. A
β442
was ignored for more than two decades, until new analytical methods
based on immunochemistry coupled with mass spectrometry revealed A
β442
as the major A
β
species
in various structures of healthy and AD brains [
3
,
4
]. A
β442
has been recently recognized as one of
Int. J. Mol. Sci. 2020,21, 9200; doi:10.3390/ijms21239200 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020,21, 9200 2 of 17
the fastest aggregating A
β
peptides and, consequently, proposed to be a key source of toxic amyloid
deposits [510].
A
β
peptides possess a number of metal-binding amino acid residues, but they are concentrated
in the N-terminal part of their sequence, including Asp1, Asp7, Glu3, Glu11, His6, His13 and His14.
On the basis of chemical and biological studies, Cu(II) ions in particular have been implicated in
the neurotoxicity of A
β
peptides. The
logK
for Cu(II) binding to A
β116
and A
β140
peptides at a
physiological pH of 7.4 was determined as 10.0 and 10.1, respectively [11].
A large volume of work was devoted to the effects of Cu(II) on the A
β
aggregation and production
of deleterious reactive oxygen species (ROS) via the Cu(II)/Cu(I) catalytic redox couple [
12
14
].
The bulk of this work was, consequently, devoted to A
β142
and A
β140
peptides, and their
C-terminally truncated models spanning the Cu(II) coordination site, A
β128
and in particular A
β116
peptides. The relevance of ROS production by the Cu(II)/Cu(I) redox process enabled by A
β1x
peptides was questioned, however, by the demonstration of a reverse relationship between the extent
of Aβ1xaggregation and toxicity and their ability to generate ROS via Cu(II) complexes [15].
On the other hand, Cu(II) complexes of A
β4x
peptides are practically redox silent, as
demonstrated in a ROS generation assay for A
β442
and its non-aggregating model A
β416
[
16
].
Moreover, the ascorbate activation ability of Cu(II)A
β416
is marginal compared to that of
Cu(II)-A
β116
[
17
]. Furthermore, Cu(II)A
β4x
form high-affinity Cu(II) complexes (log K = 13.5 and
14.2 at pH 7.4, for x = 16 and 9, respectively [
16
,
18
]). A
β416
was also able to withdraw Cu(II) ions from
A
β116
immediately and quantitatively [
16
], but, in contrast with A
β1x
peptides, strongly resisted
copper transfer to metallothionein-3, except for under highly reducing conditions [
19
,
20
]. These
properties prompted the concept that A
β442
may serve a physiological purpose in the maintenance
of synaptic transmission as a Cu(II) scavenger, as reviewed recently [
21
]. Furthermore, its hydrolytic
product, A
β49
is so inert to reduction by GSH that it may help shuttle Cu(II) ions across the
blood–brain barrier [22].
These differences between the A
β1x
and A
β4x
peptides result from the different coordination
modes they provide. The A
β1x
peptides bind the Cu(II) ion in a heterogeneous fashion, using the
N-terminal amine of Asp1, and various combinations of His6, His13 and His14 imidazole nitrogens,
and donor atoms of the Asp1-Ala2 peptide bond [
23
]. In contrast, the A
β4x
peptides take advantage
of the truncation of the first three residues, which yields the so-called ATCUN/NTS motif, generated
by Xaa-Yaa-His sequences, where the Cu(II) ion is coordinated to the N-terminal amine, the His
imidazole and two intervening peptide nitrogens [
24
26
]. Although the Jahn-Teller effect in the
d9
electronic structure of the Cu(II) ion stipulates a tetragonal symmetry with axial ligand(s), these
are usually weakly bonded when strong nitrogen ligands occupy equatorial positions. As a result,
such axial sites are often unoccupied or occupied by solvent (water) molecules, as seen in relevant X-ray
structures [
27
,
28
]. This situation has also been reproduced in the square planar coordination geometry
of the Cu(II) complex of the Phe4-Arg5-His6 ATCUN/NTS motif obtained from DFT calculations [
16
].
Taking into account the abundance of the A
β442
peptide in the brain and its possible physiological
role as a Cu(II) binding molecule, we considered that it would be very interesting to obtain a
three-dimensional structure of the A
β416
model peptide in the Cu(II)-complexed form. Unfortunately,
Cu(II) is a paramagnetic metal ion, and despite significant progress in NMR techniques, it is not
possible to obtain detailed structural data for such a small complex [29,30].
The ATCUN/NTS motif yields practically isostructural complexes for Cu(II) and all other
metal ions capable of displacing peptide nitrogens, including Ni(II) [
31
,
32
], Pd(II) and Au(III) [
33
].
These three metal ions have
d8
electronic configuration and their ATCUN/NTS complexes are
square-planar and diamagnetic. Therefore, one of these metal ions can be used to substitute for
Cu(II) in the A
β416
complex in order to perform an NMR structural study. Pd(II) is our best choice,
for its ability to form very stable peptide complexes by displacing hydrogen atoms from the side chains
as well as main-chain nitrogens even under acidic conditions [
33
,
34
]. As a consequence of the very
Int. J. Mol. Sci. 2020,21, 9200 3 of 17
high complex stability, they are also substantially more inert than the Cu(II) complexes, assuring the
slow exchange condition in NMR experiments [34].
This paper describes structural and dynamic properties of the Pd(II) complex of the
A
β416
peptide, investigated by NMR and additional techniques, including mass spectrometry,
circular dichroism, and spectrofluorimetry. We demonstrate the usefulness of Pd(II) substitution
for studies of the structure and reactivity of Cu(II) complexes of Aβ4xpeptides.
2. Results
2.1. Validation of the Pd(II) Complex as a Model for the Cu(II) Complex
Pd(II) is one of the most strongly hydrolysable cations, forming mononuclear Pd(OH)
2
species
at pH
1 and polynuclear forms at pH > 2 [
35
]. In order to avoid the kinetic entrapment of Pd(II)
in such hydroxides en route to the desired Cu(II)-A
β416
mimic, we used K
2
PdCl
4
as the Pd(II)
source, which can resist hydrolysis and polymerization up to pH 5.5 at appropriate temperatures
and concentrations [
36
38
]. In control experiments, we observed precipitation of a red-brownish
condensate after 8–10 h for samples containing 1 mM
K2PdCl4
at pH 4.5. We, therefore, adopted a
two-step preparation method of Pd(II)Aβ416.
In the first step, the peptide was incubated at room temperature with substoichiometric
(0.85 mol eq.)
K
2
PdCl
4
at pH 4.0, where the formation process of Pd(OH)
2
and its polymerization
to [Pd(OH)
2
-xClx]
n
[
39
] was slower than the anchoring of Pd(II) to the peptide. The reaction was
monitored using CD spectroscopy (Figure 1).
Figure 1.
The formation of the 1:1 Pd(II) complex of A
β416
monitored by CD. (
A
) The 40 h evolution
of CD spectra of the sample containing 0.3 mM A
β416
and 0.255 mM
K2PdCl4
at pH 4.0 and room
temperature. (
B
) The six-hour evolution of CD spectra of the same sample after the change of pH to 6.5.
(C) The time dependence of ellipticity at 360 and 321 nm.
As shown in Figure 1, the initial anchoring of the Pd(II) ion to the peptide occurred promptly,
as seen by an immediate appearance of chiral
dd
bands in the spectra at the first time point of
5 min. This initial spectrum containing at least three bands is gradually superseded by a simpler one,
Int. J. Mol. Sci. 2020,21, 9200 4 of 17
containing two distinct d-d bands. The kinetics of formation of the latter has not been completed
after 40 h. However, when NaOH was added to pH 6.5, the formation of a final reaction product was
completed within about an hour and no hydroxide precipitation was observed. Therefore, we adopted
a procedure in which the samples were incubated for 24 h at pH 4.0, followed by increasing the pH to
6.5, which was the standard value for most further experiments.
The samples prepared in this way were controlled by HPLC and major fractions were investigated
by ESI-MS. Figure 2presents an example of such data. Two major peaks contained uncoordinated
A
β416
(at 27.2 min) and a monomeric Pd(A
β416
) complex (at 37.7 min). Several minor peaks
also contained the complex of the same stoichiometry. Both the peptide and the complex exhibited
three charge species, 2+, 3+ and 4+ in electrospray ionisation mass spectrometry (ESI-MS) spectra,
with roughly similar proportions of peak intensities. Figure 2also presents the ion-mobility
spectrometry–mass spectrometry (IMS-MS) spectra of 2+ and 3+ charge species of A
β416
and
Pd(A416), showing very little difference in drift times between the peptide and its Pd(II) complex.
Figure 2.
HPLC/MS characterization of products of the two-step procedure of obtaining the Pd(A
β416
)
samples. (
A
) HPLC chromatogram of the sample containing initially 0.5 mM A
β416
and 0.425 mM
K
2
PdCl
4
. (
B
) ESI-MS spectra of the respective HPLC peaks. (
C
) IMS characterization of A
β416
and
Pd(Aβ416) species at pH 6.5.
The next experiment compared the effect of Pd(II) and Cu(II) ions on Tyr10 fluorescence, a common
tool in studying metal binding to A
β
peptide [
11
,
16
]. In order to maintain a coherence between the
Pd(II) and Cu(II) complexes, the samples were prepared in a different way. The required aliquots of
stock solutions of Cu(II) salt were added directly to individual 25
µ
M A
β416
samples dissolved in
20 mM MES buffer at pH 6.5, or 20 mM HEPES at pH 7.4. The Pd(II) samples were prepared using a
two-step procedure: initially, appropriate Pd(II) solutions with A
β416
peptide were prepared in water,
at pH 4, and incubated for 48 h. Next, the appropriate buffer was added to achieve a final solution with
20 mM MES, pH 6.5, or 20 mM HEPES, pH 7.4. The resulting titration curves, either for pH 6.5 (Figure
3) and 7.4 (Figure S1) indicate that both metal ions quench Tyr10 fluorescence in a similar stepwise
manner. The straight lines in Figure 3and Figure S1 are linear fits to the titration curve segments
Int. J. Mol. Sci. 2020,21, 9200 5 of 17
corresponding to the binding of the metal ions at the 1st and 2nd binding sites, demonstrated for Cu(II)
ions [16], and presumably equally valid for Pd(II).
Figure 3.
A
β416
Tyr10 fluorescence (
λex
= 280 nm,
λem
= 303 nm) quenching by Cu(II) (red dots) and
Pd(II) (blue circles). Regions corresponding to the binding of the first and second metal ion equivalents
are marked by dashed lines. [Aβ] = 25 µM, [MES] = 20 mM, pH 6.5.
The results of the above experiments, taken together, strongly supported the assumption that
Pd(II) may be an isostructural substitute for Cu(II) in the ATCUN/NTS motif of A
β416
for structural
studies, forming a mononuclear 1:1 species at pH 6.5 and 7.4. Therefore, we performed a series of
NMR experiments aimed at elucidating the solution structure of this complex.
2.2. Structural Analysis of the Aβ46Peptide as a Simple Model of the ATCUN/NTS Site in Aβ416
The short Phe-Arg-His-amide (FRH) peptide represents the N-terminal residues forming the
Pd(II) binding ATCUN/NTS motif. For structural and dynamic aspects of Pd(II) binding, two FRH
samples—without the metal (
apo
) and saturated with Pd(II)—were used to perform homo- and
heteronuclear NMR experiments. The acquired experimental data enabled the assignment of all
1
H
and
13
C resonances in both (
apo
and Pd(II) saturated) forms (Tables S1 and S2). The
1
H-
13
C HSQC
spectra recorded for the
apo
A
β46
and Pd(A
β46
) are presented in Figure 4. The amide
1
H
N
signals
of Arg5 and His6 were not detected due to fast exchange with water in the
apo
form, and coordination
of the Pd(II) binding in the complex. In the
1
H-
15
N HSQC spectrum, only the signals from the NH
2
group at the Phe4 N-terminus were detected (Figure S3). Large downfield chemical shifts along the
13
C axis were observed for
13
C
α
signals for Phe4 and Arg5, together with and
1
H shift for
13
C
α
1
H
α
in
His6 (Figure 4A) confirmed that backbone amide groups facilitate the coordination sites for Pd(II) ion.
The fourth site was determined from the analysis of the aromatic part of the
1
H-
13
C HSQC spectrum
(Figure 4B) and selected as
1
H
e
1 proton in His6. The geometric parameters of the FRH complex
with Pd(II) were extracted from the high-quality 3D structure of GGH tripeptide with Pd(II) ion [
33
].
Finally, the high-resolution 3D structure of FRH–Pd(II) complex (Figure 5) was solved by refined initial
structure in water box with the YASARA software [40].
Int. J. Mol. Sci. 2020,21, 9200 6 of 17
Figure 4.
Overlay of the 2D heteronuclear
1
H-
13
C HSQC NMR spectra recorded for (
A
) aliphatic and
(
B
) aromatic regions for A
β46
peptide in apo (red) and Pd(II) saturated (blue) forms. The assignments
and changes in the position of the resonances are shown. The experiments were performed on Varian
Inova 500 NMR spectrometer.
Int. J. Mol. Sci. 2020,21, 9200 7 of 17
Figure 5.
The 3D structure of Phe-Arg-His-amide (FRH) peptide represented the N-terminal
ATCUN/NTS motif saturated with Pd(II) ion based on collected NMR constraints and crystallographic
data available for GGH tripeptide [33].
To obtain information on molecular dynamic processes upon Pd(II) binding, the
13
C relaxation
rates
(R1and R2)
were measured for the
13
C resonances in Phe4 and His6 aromatic side-chains (Figure
S4). The
13
C
δ
and
13
C
e
resonances in the Phe4 side-chain did not reveal differences between the
apo and Pd(II) cases, suggesting that they were not affected by Pd(II) binding. In contrast, the
13
C
resonances in the His6 side-chain demonstrated substantial differences between both states, confirming
the formation of the coordination bond in the His6 imidazole ring. The
R2
relaxation rate for
13
C
δ2
was
significantly decreased by Pd(II). At the same time, the Pd(II) binding stimulated the increase of
R1
relaxation rate for both His6 resonances–
13
C
e1
and
13
C
δ2
(Figure S4, Table S3). Taking into account that
the
R2
relaxation rate reflects the intensity of the dynamic processes in the low-frequency time frame
(ms–
µ
s), we can conclude that Pd(II) binding resulted in shifting the molecular dynamics processes
from the ms–µs to the µs–ns regime (Figure S4A,B).
2.3. Solution Structure of the Pd(II) Complex with the Aβ416 Peptide
The A
β416
peptide together with the Pd(A
β416
) complex were subjected to structural analysis
in solution based on NMR data. The combination of homonuclear and heteronuclear NMR spectra
yielded the assignments of more than 95% of
1
H,
13
C and
15
N resonances in both forms (See Supporting
Materials, Tables S4 and S5). In fact, in the
apo
A
β416
peptide, only amide protons in the three
N-terminal residues were not observed, due to fast exchange with water protons. The resonances of
His14 were not assigned due to degeneration of signals from His13 (Table S4). For the Pd(A
β416
)
complex, the analysis of NMR data enabled us to assign practically all resonances, except for the amide
protons of Phe4, Arg5 and His6, together with the H
δ1
proton in His6, which were displaced upon
Pd(II) binding (Table S5).
Neither the A
β416
peptide nor the Pd(A
β416
) complex yielded a substantial amount of
nontrivial long- and medium-distance constraints in homonuclear NOESY or/and ROESY experiments.
Therefore, the 3D structure of
apo
A
β416
in solution was evaluated with the Xplor-NIH (version 2.39)
software mostly on the basis of backbone
φ
and
ψ
torsion angles, deduced from chemical shifts with
Int. J. Mol. Sci. 2020,21, 9200 8 of 17
the TALOS-N program [
41
]. The ensemble of 20 low-energy structures after additional refinement
with the explicit solvent model demonstrated the existence of some structuring only in the His6–Tyr10
region (Figure S6).
The 3D structure of the complex A
β416
with an equimolar amount of Pd(II) reveals metal
coordination according the ATCUN/NTS motif (Figure 6). Performed structural analysis suggest
that Pd(II) bind to A
β416
in the same manner as to the Pd(A
β46
). In comparison to the
apo
form,
the equimolar complex of Pd(II) resulted in the small chemical shift perturbations (csp) detected
for amide
1
H
N
protons for the residues in
7
DSGYEVHHQK
16
fragment of the A
β416
peptide.
There are two sets signals observed for Tyr10 (Figure S5B), which suggests this residue exists in
two conformations.
Figure 6.
The 3D structure of Pd(A
β416
) complex included the N-terminal ATCUN/NTS motif
binding the Pd(II) ion.
2.4. Translational Mobility of Aβ416 and Pd(Aβ416) in Solution
The translational mobilities of the Pd(A
β416
) complexes in solution were studied by diffusion
measurements obtained at 11.7 T. The experimental data were analyzed on the basis of the
Stejskal-Tanner equation [42]:
I=I0exp(D(Gγδ)2(δ/3))
where
γH
is the
1
H gyromagnetic ratio,
δ
is the gradient duration,
is the diffusion time and
G
is the
gradient strength. The coefficient of translational diffusion (
Dtr
) in solution was obtained for A
β416
peptide in
apo
form and for two concentrations of Pd(II) ions (Figure 7). The
Dtr
value obtained for
the
apo
-peptide is 1.64
±
0.01
×
10
10
(m
2
/s), for the equimolar ratio in the Pd(A
β416
) complex,
the measured
Dtr
was 1.65
±
0.02
×
10
10
m
2
/s which corresponds to an effective hydrodynamic
volume 7.42 (nm
3
). The Pd(A
β416
) complex 1:1.4 characterized
Dtr
equal to 1.34
±
0.02
×
10
10
m
2
/s,
corresponding to the effective hydrodynamic volume 15.98 (nm
3
), which is more than two times
higher compared to
apo
-A
β416
. The apparent hydrodynamic volume was calculated according to
the Stokes–Einstein equation using a spherical approximation. Although the morphology of the
diffusing species is not spherical, the proposal approximation is widely used to control the aggregation
phenomena [43,44].
Int. J. Mol. Sci. 2020,21, 9200 9 of 17
Figure 7.
Integral attenuation vs. gradient amplitude in PGSE NMR experiment for A
β416
and
Pd(A
β416
) complex in solution. The
apo
A
β416
peptide (filled circles), equimolar Pd(A
β416
)
complex 1:1 (open circles) and 1:1.4 (open squares). The experiments were performed on a Varian Inova
500 NMR spectrometer.
3. Discussion
The CD and fluorescence spectroscopic and MS data strongly suggested that the Pd(II) ion
accommodated the same ATCUN/NTS coordination structure in A
β416
as Cu(II). The evidence was
indirect, however. For CD (Figure 1), it was based on the characteristic alternate pattern of Pd(II)
dd
bands, blueshifted, compared to the Cu(II) case, but retaining their symmetry [
16
]. For Tyr10
fluorescence, the same quenching pattern was seen for both metal ions (Figure 3), with the quenching
just slightly more effective for the first Pd(II) equivalent. The correct stoichiometry indicating the
replacement of four hydrogens by Pd(II) coordination was provided by ESI-MS (Figure 2A). In addition,
the overlapping of the IMS drift peaks for the apo peptide and the complex is consistent with this
view, because the lack of drift time difference indicates that the Pd(II) ion did not produce the
long-range structuring in the molecule, and hence it had to be coordinated locally at one end of the
molecule (Figure 2B).
These observations prompted the NMR study of the structure and dynamics of the Pd(A
β416
)
complex. However, while the observations presented above strongly suggested that the Pd(II) ion
was bound selectively at the ATCUN/NTS Phe4-Arg5-His6 sequence, some binding at His13 and/or
His14 residues could not be excluded a priori. Therefore, structural experiments were also performed
using the Phe-Arg-His-amide tripeptide. This peptide has essentially one possible Pd(II) binding
mode at weakly acidic pH, namely the ATCUN/NTS four-nitrogen complex, as evidenced by the
X-ray study of its analogue Gly-Gly-His [
33
]. It could, therefore, serve as a positive control for the
A
β416
experiments. The analysis of NMR spectra of the tripeptide complex, presented in Figures 4
and S3 confirmed the expected coordination mode. Very interestingly, the Pd(II) complexation did not
affect the dynamics of the Phe4 side chain, but significantly elevated the longitudinal and decreased
the transverse
13
C relaxation rates for the His imidazole ring. This observation is in line with the
formation of the six-membered chelate ring formed by simultaneous coordination of His amide and
His N1 nitrogens.
The experiments performed for the equimolar Pd(A
β416
) sample confirmed the Pd(II)
coordination in the 4N mode to the Phe-Arg-His sequence, and the lack of longer distance structuring
in the coordinated peptide, as shown in Figure 6. In particular, there was no interaction between Tyr10
Int. J. Mol. Sci. 2020,21, 9200 10 of 17
and the Pd(II) coordination site, which was previously detected for a generally similar Ni(II) complex
with the N-terminal peptide of HP2 protamine [
45
]. In the structure of the Pd(II) binding site in the
tripeptide Phe-Arg-His (A
β46
) presented in Figure 5, no interactions between the Phe, Arg and His
side chains are present. These side chains are located away from each other. In particular, there is no
axial interaction of Phe4
π
charge with the axial electronic density of the Pd(II). Such interaction was
seen before in the NMR structures of di- and tripeptides containing a Tyr residue [
46
,
47
], but apparently
is not sufficiently effective for the less polarized Phe aromatic ring. The positively charged guanidinium
group of Arg5 appears to be fixed in its position by electrostatic interaction with the partial negative
charge located on Pd(II)-coordinated amide nitrogens [
45
]. The Phe4 and Arg5 positions are also similar
to those calculated previously for the Cu(II) complex with the A
β47
fragment by DFT [
16
]. The same
structure was retained in Pd(A
β416
). The hydrogen bond between Phe4 and Asp7 postulated by
previous DFT calculations was not detected.
The mobility of Pd(A
β416
) was studied in comparison to the
apo
-peptide, and also in the presence
of the
apo
-peptide excess. Interesting observations were made in these experiments. In agreement
with the IMS data, the Pd(II) coordination did not affect the peptide’s hydrodynamic radius, thereby
confirming the purely local character of the complexation on the peptide structure. A significant
decrease in the complex mobility was observed, however, in the sample containing the complex
and a 40% excess of the peptide. The apparent hydrodynamic radius calculated for this sample was
about two-times larger than that for either the
apo
-peptide or the complex. This effect can be only
explained by the supramolecular assembly between the complex and the
apo
-peptide. The absence
of changes in chemical shifts of the Phe-Arg-His sequence upon
apo
-peptide addition indicates
that no coordinative bridging by Pd(II) occurred. Two possible mechanisms for this effect can be
considered. As the square-planar Pd(II) chelate structure is the only permanent and significant
difference between the complexed peptide and the
apo
-peptide, one possible interaction involves
its dipolar interaction with aromatic residues of the
apo
-peptide, similar to the interaction observed
between the isoelectronic Ni(II) complex of the Arg-Thr-His N-terminus and the Tyr phenol ring
in the HP2 pentadecapeptide [
45
]. In general, many types of stacking interactions that have been
observed in various Cu(II) and Pd(II) biomimetic complexes might occur in the investigated system [
48
].
Another may stem from electrostatics, as proposed recently in studies of Cu(II) effects on fibrillization
of A
β140
and A
β440
peptides [
49
]. The electrostatic charges of A
β416
and Pd(A
β416
) at pH 6.5 can
be calculated from the data presented before for the Cu(II) complex, reasonably assuming that the
Cu(II)/Pd(II) replacement did not affect the acidities of the peptide’s residues. The average charges
of these two species are +1.1 and +0.5, respectively. If, however, we consider the +1 charge at the
C-terminal Lys residue in both molecules, then we see that there is a slight electrostatic incentive for
the attractive interaction specifically between the
apo
-peptide and the complex spread over the rest of
the molecule. Probably, both kinds of interactions occur, enabling the formation of the heterodimer or
even higher order assemblies, even despite the lack of defined structure in free and Pd(II)-complexed
A
β416
. These results confirm the suitability of Pd(II) substitution to study structural aspects of Cu(II)
complexes of A
β
peptides and suggest that the molecular pathway of their aggregation processes may
lead via interactions of their N-termini.
4. Materials and Methods
4.1. Materials
The A
β416
(FRHDSGYEVHHQK-amide) and A
β46
(FRH-amide) peptides were synthesized
according to Fmoc strategy as described previously [
16
]. K
2
PdCl
4
, NaOH, HCl and acetonitrile (HPLC
grade) were purchased from Sigma-Aldrich. D2O was purchased from Armar Chemicals.
Int. J. Mol. Sci. 2020,21, 9200 11 of 17
4.2. Sample Preparation
A portion of lyophilized A
β416
peptide was diluted in MQ water. The concentration of this
stock solution was determined using
e275
= 1375 M
1
cm
1
[
16
]. Pd(II) was added to the sample from
a 100 mM K
2
PdCl
4
stock of to obtain a peptide-to-metal ratio of 1:0.85. The pH of the sample was
then increased by adding small amounts of concentrated NaOH up to pH 4, and then handled further,
as required by specific experimental methods.
4.3. Mass Spectrometry
The samples prepared as above, containing 0.5 mM A
β416
and the peptide-to-Pd(II) ratio of
1:0.85 were incubated for a further 24 h at pH 4. Then, the pH was set to 6.5 using concentrated
NaOH. 100
µ
L samples were injected into the HPLC system (Empower, Waters), equipped with an
analytical C18 column (4.6
×
250 mm). The eluting solvent A was 0.1% (v/v) TFA in water, and solvent
B was 0.1% (v/v) TFA in 90% (v/v) acetonitrile. The chromatograms were obtained at 220 and 280 nm.
Individual peaks were collected and measured by ESI-MS on a ESI Q-ToF Premier mass spectrometer
(Waters). The samples were injected at a 40 mL/min flow rate and MS spectra were recorded in positive
ion mode during 5 min in the range
m/z
of 500–1800. Obtained mass spectrometry data were analyzed
and processed using MassLynx (Version 4.1, Waters Inc., Milford, MA, USA). Ion mobility (IMS-MS)
experiments were performed using a Synapt G2 HDMS instrument (Waters). Ions were generated
using nanoelectrospray ionization at 1.7 kV from PicoTip emitters 2
µ
m i.d. (QT10-70-2-CE-20 New
Objective). MS settings were adjusted to obtain an optimal ion transmission as follows: 30 V sampling
cone, 5 V extractor cone, 40
C source temperature, 10 V trap collision energy, and 5 V transfer
collision energy, wave height and wave velocity were set as 40 V and 800 m/s, respectively. Drift times
were obtained by generating an extracted ion chromatogram (XIC) from the arrival time distribution
function in MassLynx v4.1 using the monoisotopic mass and a mass window of ±0.075 Da.
4.4. Circular Dichroism
CD experiments were carried out on the J-815 CD spectrometer (JASCO) over the spectral range
of 250–600 nm, using a 1 cm path length quartz cuvettes. Measurements were performed at 25
C
for samples containing 0.3 mM A
β416
and the peptide-to-Pd(II) ratio of 1:0.85. The spectra were
recorded in 40 min intervals, starting immediately after sample preparation, until the reaction neared
equilibrium after 40 h. Next, the pH was set to 6.5 using NaOH and spectral changes were recorded at
30 min intervals for another six hours.
4.5. Spectrofluorimetry
Fluorescence spectra of A
β416
in the presence of Cu(II) and Pd(II) ions were recorded at 25
C
using a FP-6500 spectrofluorometer (Jasco). The excitation wavelength was 280 nm; the emission
spectra were in the range of 290–400 nm. Solutions of metal ions and the peptide were combined in
varying metal/peptide ratios, with a constant A
β416
concentration of 25
µ
M, in 20 mM MES buffer,
pH 6.5, or 20 mM HEPES buffer, pH 7.4. Cu(II) salt solution was added directly to the prepared
peptide solution in the respective buffer. Pd(II) was added to water solutions of the peptide at pH 4,
then incubated for 48 h. Next, to such solutions, appropriately concentrated buffer solutions were
added to obtain concentrations analogous to Cu(II) samples. Each sample was prepared in triplicate.
4.6. NMR Spectroscopy
The Pd(II)-containing samples were prepared by incubation for 24 h at room temperature at
pH 4.0
. Next, the samples were diluted with D
2
O to obtain a 10% (v/v) solution of the latter,
followed by adjustment of pH to 6.5 with a small amount of concentrated NaOH. The samples of the
apo A
β46
and A
β416
peptide was prepared directly in 90%/10% (v/v) H
2
O/D
2
O, at pH 6.5. The final
volumes of the samples were 300 µL, with final peptide concentrations between 1.0 and 2.5 mM.
Int. J. Mol. Sci. 2020,21, 9200 12 of 17
The measurements were conducted on Agilent DDR2 800 (
1
H frequency 799.903 MHz), Agilent
DDR2 600 (
1
H frequency 599.930 MHz), and Varian Inova 500 (
1
H frequency 500.606 MHz) NMR
spectrometers operated at magnetic fields of 18.8 T, 14.1 T, and 11.7 T, respectively. All spectrometers
were equipped with three channels,
z
-gradient unit and
1
H/
13
C/
15
N probehead with inverse detection.
The homonuclear 2D NMR experiments included TOCSY acquired with mixing times of 15, 80,
and 90 ms, ROESY conducted with the mixing time of 300 ms, and NOESY with mixing times of
150 and 300 ms. Homonuclear experimental data sets were supplemented with heteronuclear 2D
1
H-
13
C HSQC (tuned independently to aliphatic and aromatic regions) as well as 2D
1
H-
15
N HSQC
NMR experiments acquired using the natural abundance of
13
C and
15
N isotopes. All NMR data
sets were referenced indirectly in respect to external sodium 2,2-dimethyl-2-silapentane-5-sulfonate
(DSS) with the
Ξ
coefficients equal to 0.251449530 and 0.101329118 for
13
C and
15
N dimensions,
respectively [
50
]. The acquired data sets were processed with the NMRPipe program [
51
] and analyzed
with the Sparky software [52].
4.7. Assignment of the 1H, 13C and 15N Resonances and 3D Structure Evaluation of Aβ416 Peptide and
Pd(Aβ416) Complex in Solution
The
1
H,
13
C and
15
N resonance assignments were obtained using base standard procedure on
base analysis 2D homonuclear (NOESY, TOCSY) and heteronuclear (
1
H-
13
C and
1
H-
15
N HSQC)
spectra. More than 90% of resonances were successfully assigned (Tables S4 and S5). Unfortunately,
the collected 2D NOESY and ROESY data sets did not provide nontrivial medium- or long-range
distance constraints. Nevertheless, the analysis of
1
H,
13
C and
15
N chemical shifts performed with
the TALOS-N software [
41
] yielded 16 and 8 backbone
φ
and
ψ
torsion angles predicted for A
β416
peptide in apo and Pd(II)-saturated forms, respectively. Additionally, the
χ
1 torsion angles of the side
chains were predicted in six and four residues in A
β416
peptide and Pd(A
β416
) complex, respectively
(Tables S6 and S7). Both 3D structures were solved by the Xplor-NIH 2.37 program [
53
]. The slightly
modified standard protocol included in Xplor-NIH distribution (protG.inp) was used for 3D structure
evaluation. Briefly, the 200 randomly generated structures which were subjected to 12 ns cartesian
molecular dynamic simulation at 2000 K followed by 3000 steps of simulating annealing included slow
cooling to the temperature 100 K. The refined procedure in the explicit solvent were performed for
the 20 lowest-energy structures with the YASARA software utilizing AMBER14 force field during the
3000 steps of simulating annealing procedure in water solvent [
40
]. In the case of Pd(A
β416
) complex,
the parameters for Pd(II) ion (bond length and angles) were taken from the crystal structure of Pd(II)
complex with the GGH tripeptide [
33
]. The evaluated 3D structures were visualized and analyzed
with the MOLMOL [54] and Chimera [55] software.
4.8. 13C Relaxation Measurements
The
13
C
R1
and
R2
relaxation data were acquired on Varian Inova 500 NMR spectrometer for
FRH peptide in both (apo and Pd(II) saturated) at natural abundance of
13
C isotope. The experiments
were conducted utilizing the pulse sequence included in BioPack (Agilent Inc., PaloAlto, CA, USA).
The 32 scans were enabled to achieve a reasonable signal-to-noise ratio for collected points in
R1
and
R2
measurements. The
R1
relaxation data were obtained as 5 delays—10, 50, 110, 190, and 290 ms.
The
R2
relaxation rates were recorded with 5 delays—10, 30, 50, 70, and 90 ms. The recycling delay
was set to 3 s.
4.9. Diffusion Measurements
Diffusion experiments were carried out on a Varian Inova 500 NMR spectrometer utilizing
a standard PGSE (Pulsed Gradient Spin Echo) pulse sequence [
56
] supplemented with Excitation
sculpting Solvent Suppression block [
57
] were applied as 25 gradients with an effective gradient
pulse duration (
δ
) as long as 3 ms. The diffusion measurements for A
β416
saturated with Pd(II)
were performed using diffusion time (
) of 100 and 150 ms in the case of Pd(II) concentrations 1
Int. J. Mol. Sci. 2020,21, 9200 13 of 17
and 1.4. The 128 accumulations were performed with a relaxation delay of 3 s in order to increase
the signal-to-noise ratio. The obtained experimental data were Fourier transformed with a 2 Hz line
broadening factor applied. For extraction of the translation diffusion coefficient (
Dtr
), the signals
observed between 0–3 ppm were integrated and then exported to the Origin software together with
gradient amplitudes. The
Dtr
values were extracted by fitting using the Stejskal–Tanner equation [
42
]
taking into account an additional correction for the
delay during BPP pulse in sequence (delay
between gradient δand π/2 pulse was equal to 0.5 ms).
5. Conclusions
Our study demonstrated that by Pd(II) substitution working models of Cu(II) complexes of
ATCUN/NTS peptides, including those of the A
β4x
family, suitable for direct structural and
functional studies can be obtained. Already in this pioneering study, the diffusion coefficient
determination, essentially impossible for the paramagnetic Cu(II) complex empowered us to confirm
a supramolecular interaction contributing to the control of aggregation and fibrillization of the A
β
peptides. This interaction is a prerequisite for a better understanding of the molecular events leading
to Alzheimer’s disease and thereby finding key markers of the disease.
Supplementary Materials:
The following are available online at http://www.mdpi.com/1422-0067/21/23/9200/
s1 Table S1:
1
H, and
13
C chemical shifts assigned for the
apo
A
β46
peptide at 298 K on Varian Inova 500 NMR
spectrometer. Table S2:
1
H, and
13
C chemical shifts assigned for the Pd(A
β46
) complex at 298 K on Varian
Inova 500 NMR spectrometer. Table S3: The
13
C
R1
and
R2
relaxation rates extracted for the aromatic carbons
in the FRH peptide acquired on natural abundance of
13
C isotope. The NMR experiment performed at 298 K
on Varian Inova 500 NMR spectrometer. Table S4:
1
H,
13
C, and
15
N chemical shifts assigned for apo A
β416
peptide at 298 K on Agilent DDR2 800 NMR spectrometer. Table S5:
1
H,
13
C, and
15
N chemical shifts assigned
for Pd(A
β416
) complex at 298 K on Agilent DDR2 800 NMR spectrometer. Table S6: The restrains for
ψ
and
φ
backbone and
χ1
side-chain torsion angles evaluated by TALOS-N program for apo A
β416
peptide on base
1
H,
13
C, and
15
N chemical shifts. Table S7: The restrains for
ψ
and
φ
backbone and
χ1
side-chain torsion angles
evaluated by TALOS-N program for Pd(A
β416
) peptide on base
1
H,
13
C, and
15
N chemical shifts. Figure S1:
A
β416
Tyr10 fluorescence (
λex
= 280 nm,
λem
= 303 nm) quenching by Cu(II) (red dots) and Pd(II) (blue circles).
Regions corresponding to the binding of the first and second metal ion equivalent are marked by dashed lines.
[A
β
] = 25
µ
M, [HEPES] = 20 mM, pH 7.4 Figure S2: A
β4˘16
Tyr10 fluorescence (
λex
= 280 nm,
λem
= 290-400 nm)
quenching by Cu(II) (
A
,
C
) and Pd(II) (
B
,
D
). The changes were observed at pH 6.5 (20 mM MES,
A
,
B
) and 7.4 (20
mM HEPES,
C
,
D
). Shown are the spectra of the peptide with increasing concentrations of Cu(II) or Pd(II) ions.
The concentration of A
β4˘16
was constant (25
µ
M), and the concentrations of metal ions were as follows: 0, 4,
8, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 40, 44, 52, 60, 80, and 100
µ
M. Figure S3: The
1
H-
15
N HSQC spectrum
acquired for Pd(A
β46
) complex at 298 K. The experiments were performed on natural abundance of the
15
N
isotope Varian Inova 500 NMR spectrometer. Figure S4: The values of (
A
) longitudinal (
R1
) and (
B
) transverse (
R2
)
13
C relaxation rates measured for aromatic carbons in Phe4 and His6 in the A
β46
peptide for
apo
(red) and Pd(II)
(blue) forms. The examples of fit relaxation data for His6
13
C
δ2
in A
β46
in complex with Pd(II) presented on
panels (
C
,
D
) for
13
C
R1
and
R2
relaxation rates, respectively. The measurements were performed on the natural
abundance of
13
C isotope utilizing on Varian Inova 500 NMR spectrometer. Figure S5: The amide-aliphatic part of
homonuclear 2D
1
H-
1
H TOCSY spectra for the A
β416
peptide acquired with a 80 ms mixing time for
apo
(
A
)
and Pd(A
β416
) saturated (
B
) forms on an Agilent DDR2 800 NMR spectrometer at 293 K. The assignments in
both forms are presented as one-letter code and sequence number. In the case of the Pd(A
β416
) saturated form,
signals (Asp7, Ser8, Gly9, Tyr10) representing the
apo
A
β416
peptide are clearly visible. The whole assignments
yielded by the analysis of NMR data are presented in Tables S4 and Table S5 for the
apo
and Pd(A
β416
) form,
respectively. Figure S6: Ensemble of 20 low-energy structures of A
β416
peptide in
apo
form evaluated on the
base NMR data. The structures are fitted on central
6
HSGY
10
motif. Orientation side-chains of the His6, Asp7,
Ser8 and Tyr10 are shown in green.
Author Contributions:
Conceptualization, W.B. and T.F.; methodology, S.C.D., J.P., T.F., K.S., and I.Z.; software,
K.S., J.P., and I.Z.; validation, J.P., I.Z., T.F., and W.B.; investigation, M.M., K.S., P.S., J.P., I.Z., and T.F.; resources,
S.C.D., T.F., K.S., and W.B.; data curation, M.M., K.S., K.B.-A., P.S., T.F., I.Z.; writing–original draft preparation, T.F.,
I.Z. and W.B.; writing–review and editing, W.B.; visualization, J.P., I.Z., T.F.; supervision, W.B.; funding acquisition,
M.M., and W.B. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by Polish National Science Centre grant Preludium number
2014/13/N/ST5/01553 tp M.M.
Acknowledgments:
Authors thank to David Plonka for help with the synthesis of the A
β416
and FRH peptides.
Int. J. Mol. Sci. 2020,21, 9200 14 of 17
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
The following abbreviations are used in this manuscript:
CD Circular dichroism
ESI-MS ElectroSpray Ionisation Mass Spectrometry
IMS-MS Ion-Mobility Spectrometry Mass Spectrometry
DSS sodium 2,2-dimethyl-2-silapentane-5-sulfonate
PGSE Pulsed Gradient Spin Echo
NOESY Nuclear Overhauser SpectroscopY
HSQC Heteronuclear Single Quantum Correlation spectroscopy
TOCSY TOtal Correlation SpectroscopY
ROS Reactive Oxygen Species
AD Alzheimer’s Disease
References
1.
Masters, C.L.; Simms, G.; Weinman, N.A.; Multhaup, G.; McDonald, B.L.; Beyreuther, K. Amyloid plaque
core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad. Sci. USA 1985,82, 4245–4249.
2.
Masters, C.L.; Multhaup, G.; Simms, G.; Pottgiesser, J.; Martins, R.; Beyreuther, K. Neuronal origin of a
cerebral amyloid: Neurofibrillary tangles of Alzheimer’s disease contain the same protein as the amyloid of
plaque cores and blood vessels. EMBO J. 1985,4, 2757–2763.
3.
Lewis, H.; Beher, D.; Cookson, N.; Oakley, A.; Piggott, M.; Morris, C.; Jaros, E.; Perry, R.; Ince, P.; Kenny, R.;
et al. Quantification of Alzheimer pathology in ageing and dementia: age-related accumulation of amyloid-
β
(42) peptide in vascular dementia. Neuropathol. Appl. Neurobiol. 2006,32, 103–118.
4.
Portelius, E.; Bogdanovic, N.; Gustavsson, M.K.; Volkmann, I.; Brinkmalm, G.; Zetterberg, H.; Winblad, B.;
Blennow, K. Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and
sporadic Alzheimer’s disease. Acta Neuropathol. 2010,120, 185–193.
5.
Bouter, Y.; Dietrich, K.; Wittnam, J.L.; Rezaei-Ghaleh, N.; Pillot, T.; Papot-Couturier, S.; Lefebvre, T.; Sprenger,
F.; Wirths, O.; Zweckstetter, M.; et al. N-truncated amyloid
β
(A
β
) 4-42 forms stable aggregates and induces
acute and long-lasting behavioral deficits. Acta Neuropathol. 2013,126, 189–205.
6.
Wirths, O.; Walter, S.; Kraus, I.; Klafki, H.W.; Stazi, M.; Oberstein, T.J.; Ghiso, J.; Wiltfang, J.; Bayer, T.A.;
Weggen, S. N-truncated A
β
4–x peptides in sporadic Alzheimer’s disease cases and transgenic Alzheimer
mouse models. Alzheimers Res. Ther. 2017,9, 80.
7.
Cabrera, E.; Mathews, P.; Mezhericher, E.; Beach, T.G.; Deng, J.; Neubert, T.A.; Rostagno, A.; Ghiso,
J. A
β
truncated species: Implications for brain clearance mechanisms and amyloid plaque deposition.
Biochim. Biophys. Acta BBA Mol. Basis Dis. 2018,1864, 208–225.
8.
Wirths, O.; Zampar, S.; Weggen, S. N-Terminally Truncated A
β
Peptide Variants in Alzheimer’s Disease.
Exon Publ. 2019, 107–122, doi:10.15586/alzheimersdisease.2019.ch7.
9.
Zampar, S.; Klafki, H.W.; Sritharen, K.; Bayer, T.A.; Wiltfang, J.; Rostagno, A.; Ghiso, J.; Miles, L.A.;
Wirths, O. N-terminal heterogeneity of parenchymal and vascular amyloid-
β
deposits in Alzheimer‘s disease.
Neuropathol. Appl. Neurobiol. 2020,46, 673–685.
10.
Wirths, O.; Zampar, S. Emerging roles of N-and C-terminally truncated A
β
species in Alzheimer’s disease.
Expert Opin. Ther. Targets 2019,23, 991–1004.
11.
Alies, B.; Renaglia, E.; Rózga, M.; Bal, W.; Faller, P.; Hureau, C. Cu(II) affinity for the Alzheimer’s peptide:
Tyrosine fluorescence studies revisited. Anal. Chem. 2013,85, 1501–1508.
12.
Drew, S.C.; Barnham, K.J. The heterogeneous nature of Cu
2+
interactions with Alzheimer’s amyloid-
β
peptide. Accounts Chem. Res. 2011,44, 1146–1155.
13.
Arrigoni, F.; Prosdocimi, T.; Mollica, L.; De Gioia, L.; Zampella, G.; Bertini, L. Copper reduction and dioxygen
activation in Cu–amyloid beta peptide complexes: Insight from molecular modelling. Metallomics
2018
,
10, 1618–1630.
Int. J. Mol. Sci. 2020,21, 9200 15 of 17
14.
Atrián-Blasco, E.; del Barrio, M.; Faller, P.; Hureau, C. Ascorbate oxidation by Cu (amyloid-
β
) complexes:
Determination of the intrinsic rate as a function of alterations in the peptide sequence revealing key residues
for reactive oxygen species production. Anal. Chem. 2018,90, 5909–5915.
15.
Huang, H.; Lou, X.; Hu, B.; Zhou, Z.; Chen, J.; Tian, Y. A comprehensive study on the generation of reactive
oxygen species in Cu-Aβ-catalyzed redox processes. Free Radic. Biol. Med. 2019,135, 125–131.
16.
Mital, M.; Wezynfeld, N.E.; Fr ˛aczyk, T.; Wiloch, M.Z.; Wawrzyniak, U.E.; Bonna, A.; Tumpach, C.;
Barnham, K.J.; Haigh, C.L.; Bal, W.; et al. A functional role for A
β
in metal homeostasis? N-truncation and
high-affinity copper binding. Angew. Chem. 2015,127, 10606–10610.
17.
Esmieu, C.; Ferrand, G.; Borghesani, V.; Hureau, C. N-truncated A
β
peptides impact on Cu and Cu
(Aβ)-generated ROS: Cu(I) matters! Chem. Eur. J. 2020,26, doi: 10.1002/chem.202003949.
18.
Bossak-Ahmad, K.; Mital, M.; Płonka, D.; Drew, S.C.; Bal, W. Oligopeptides generated by neprilysin
degradation of
β
-amyloid have the highest Cu(II) affinity in the whole A
β
family. Inorg. Chem.
2018
,
58, 932–943.
19.
Wezynfeld, N.E.; Stefaniak, E.; Stachucy, K.; Drozd, A.; Płonka, D.; Drew, S.C.; Kr˛e˙
zel, A.; Bal, W. Resistance
of Cu (A
β
4–16) to Copper Capture by Metallothionein-3 Supports a Function for the A
β
4–42 Peptide as a
Synaptic CuII Scavenger. Angew. Chem. Int. Ed. 2016,55, 8235–8238.
20.
Santoro, A.; Wezynfeld, N.E.; Vašák, M.; Bal, W.; Faller, P. Cysteine and glutathione trigger the Cu–Zn swap
between Cu(II)-amyloid-
β
4-16 peptide and Zn 7-metallothionein-3. Chem. Commun.
2017
,53, 11634–11637.
21.
Stefaniak, E.; Bal, W. CuII Binding Properties of N-Truncated A
β
Peptides: In Search of Biological Function.
Inorg. Chem. 2019,58, 13561–13577
22.
Stefaniak, E.; Płonka, D.; Szczerba, P.; Wezynfeld, N.E.; Bal, W. Copper Transporters? Glutathione Reactivity
of Products of Cu–AβDigestion by Neprilysin. Inorg. Chem. 2020,59, 4186–4190.
23.
Atrián-Blasco, E.; Gonzalez, P.; Santoro, A.; Alies, B.; Faller, P.; Hureau, C. Cu and Zn coordination to
amyloid peptides: From fascinating chemistry to debated pathological relevance. Coord. Chem. Rev.
2018
,
371, 38–55.
24.
Sigel, H.; Martin, R.B. Coordinating properties of the amide bond. Stability and structure of metal ion
complexes of peptides and related ligands. Chem. Rev. 1982,82, 385–426.
25.
Harford, C.; Sarkar, B. Amino terminal Cu (II)-and Ni (II)-binding (ATCUN) motif of proteins and peptides:
Metal binding, DNA cleavage, and other properties. Accounts Chem. Res. 1997,30, 123–130.
26.
Gonzalez, P.; Bossak, K.; Stefaniak, E.; Hureau, C.; Raibauta, L.; Bal, W.; Faller, P. N-terminal Cu binding
motifs Xxx-Zzz-His (ATCUN) and Xxx-His and their derivatives: Chemistry, biology and medicinal
applications. Chem. Eur. J. 2018,24, 8029–8041.
27.
Hureau, C.; Eury, H.; Guillot, R.; Bijani, C.; Sayen, S.; Solari, P.L.; Guillon, E.; Faller, P.; Dorlet, P. X-ray
and Solution Structures of Cu
II
GHK and Cu
II
DAHK Complexes: Influence on Their Redox Properties.
Chem. Eur. J. 2011,17, 10151–10160.
28.
Camerman, N.; Camerman, A.; Sarkar, B. Molecular design to mimic the copper (II) transport site of
human albumin. The crystal and molecular structure of copper (II)–glycylglycyl-L-histidine-N-methyl amide
monoaquo complex. Can. J. Chem. 1976,54, 1309–1316.
29.
Donaldson, L.W.; Skrynnikov, N.R.; Choy, W.Y.; Muhandiram, D.R.; Sarkar, B.; Forman-Kay, J.D.;
Kay, L.E. Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation
enhancement NMR spectroscopy. J. Am. Chem. Soc. 2001,123, 9843–9847.
30.
Nair, N.G.; Perry, G.; Smith, M.A.; Reddy, V.P. NMR studies of zinc, copper, and iron binding to histidine,
the principal metal ion complexing site of amyloid-βpeptide. J. Alzheimer’s Dis. 2010,20, 57–66.
31.
Bal, W.; Djuran, M.I.; Margerum, D.W.; Gray, E.T.; Mazid, M.A.; Tom, R.T.; Nieboer, E.; Sadler, P.J.
Dioxygen-induced decarboxylation and hydroxylation of [NiII (glycyl-glycyl-L-histidine)] occurs via Ni
III: X-ray crystal structure of [NiII (glycyl-glycyl-
α
-hydroxy-D,L-histamine)]
·
3 H
2
O. J. Chem. Soc. Chem.
Commun. 1994,16, 1889–1890.
32.
Bal, W.; Chmurny, G.N.; Hilton, B.D.; Sadler, P.J.; Tucker, A. Axial hydrophobic fence in highly-stable Ni(II)
complex of des-angiotensinogen N-terminal peptide. J. Am. Chem. Soc. 1996,118, 4727–4728.
33.
Best, S.L.; Chattopadhyay, T.K.; Djuran, M.I.; Palmer, R.A.; Sadler, P.J.; Sóvágó, I.; Varnagy, K. Gold(III) and
Palladium(II) Complexes of Glycylglycyl-L-Histidine: Crystal Structures of [AuIII (Gly-Gly-L-His-H
2
)]
Cl
·
H
2
O and [PdII (Gly-Gly-L-His-H
2
)]
·
1.5 H
2
O and His
ε
NH deprotonation. J. Chem. Soc. Dalton Trans.
1997, 2587–2596.
Int. J. Mol. Sci. 2020,21, 9200 16 of 17
34.
Klein, A.; Tsiveriotis, P.; Malandrinos, G.; Hadjiliadis, N. Platinum (II) and palladium (II) complexes with
histidine and histidyl containing peptides: Structure and reactivity. Rev. Inorg. Chem. 2000,20, 305–338.
35.
Frias, E.C.; Pitsch, H.; Ly, J.; Poitrenaud, C. Palladium complexes in concentrated nitrate and acid solutions.
Talanta 1995,42, 1675–1683.
36.
van Middlesworth, J.M.; Wood, S.A. The stability of palladium (II) hydroxide and hydroxy–chloride
complexes: An experimental solubility study at 25–85 C and 1 bar. Geochim. Cosmochim. Acta
1999
,
63, 1751–1765.
37.
Mili´c, N.B.; Bugarˇci´c, Ž.D. Hydrolysis of the palladium (II) ion in a sodium chloride medium.
Transit. Met. Chem. 1984,9, 173–176.
38.
Torapava, N.; Elding, L.I.; Mändar, H.; Roosalu, K.; Persson, I. Structures of polynuclear complexes of
palladium (II) and platinum (II) formed by slow hydrolysis in acidic aqueous solution. Dalton Trans.
2013
,
42, 7755–7760.
39.
Boily, J.F.; Seward, T.M.; Charnock, J.M. The hydrolysis and precipitation of Pd (II) in 0.6 mol kg- 1 NaCl: A
potentiometric, spectrophotometric, and EXAFS study. Geochim. Cosmochim. Acta 2007,71, 4834–4845.
40.
Krieger, E.; Koraimann, G.; Vriend, G. Increasing the precision of comparative models with YASARA
NOVA–a self-parameterizing force field. Proteins Struct. Funct. Bioinform. 2002,47, 393–402.
41.
Shen, Y.; Bax, A. Protein structural information derived from NMR chemical shift with the neural network
program TALOS-N. In Artificial Neural Networks; Springer: Berlin/Heidelberg, Germany, 2015; pp. 17–32.
42.
Stejskal, E.O.; Tanner, J.E. Spin diffusion measurements: Spin echoes in the presence of a time-dependent
field gradient. J. Chem. Phys. 1965,42, 288–292.
43.
Macchioni, A.; Ciancaleoni, G.; Zuccaccia, C.; Zuccaccia, D. Determining accurate molecular sizes in solution
through NMR diffusion spectroscopy. Chem. Soc. Rev. 2008,37, 479–489.
44.
Taube, M.; Pietralik, Z.; Szymanska, A.; Szutkowski, K.; Clemens, D.; Grubb, A.; Kozak, M. The domain
swapping of human cystatin C induced by synchrotron radiation. Sci. Rep. 2019,9, 8548.
45.
Bal, W.; Wójcik, J.; Maciejczyk, M.; Grochowski, P.; Kasprzak, K.S. Induction of a secondary structure in
the N-terminal pentadecapeptide of human protamine HP2 through Ni(II) coordination. An NMR study.
Chem. Res. Toxicol. 2000,13, 823–830.
46.
Kozłowski, H. Spectroscopic and magnetic resonance studies on Ni(II), Cu(II) and Pd(II) complexes with
Gly-Leu-Tyr and Tyr-Gly-Gly tripeptides. Inorganica Chim. Acta 1978,31, 135–140.
47.
Kozłowski, H.; Je˙
zowska, M.; Szyszuk, H. PMR conformational studies of Pd(II) complexes with Ala-Tyr
and d-Leu-Tyr depeptides. J. Mol. Struct. 1978,50, 73–80.
48.
Yamauchi, O. Noncovalent interactions in biocomplexes. Phys. Sci. Rev.
2016
,1, doi:10.1515/psr-2016-0001.
49.
Stefaniak, E.; Atrian-Blasco, E.; Goch, W.; Sabater, L.; Hureau, C.; Bal, W. The aggregation pattern of A
β
1-40
is altered by the presence of N-truncated A
β
4-40 and/or Cu(II) ions in a similar way via ionic interactions.
Chem. Eur. J. 2020, in press.
50.
Wishart, D.S.; Bigam, C.G.; Yao, J.; Abildgaard, F.; Dyson, H.J.; Oldfield, E.; Markley, J.L.; Sykes, B.D.
1
H,
13
C
and 15N chemical shift referencing in biomolecular NMR. J. Biomol. NMR 1995,6, 135–140.
51.
Delaglio, F.; Grzesiek, S.; Vuister, G.W.; Zhu, G.; Pfeifer, J.; Bax, A. NMRPipe: A multidimensional spectral
processing system based on UNIX pipes. J. Biomol. NMR 1995,6, 277–293.
52.
Lee, W.; Tonelli, M.; Markley, J.L. NMRFAM-SPARKY: Enhanced software for biomolecular NMR
spectroscopy. Bioinformatics 2015,31, 1325–1327.
53.
Schwieters, C.D.; Kuszewski, J.J.; Clore, G.M. Using Xplor–NIH for NMR molecular structure determination.
Prog. Nucl. Magn. Reson. Spectrosc. 2006,48, 47–62.
54.
Koradi, R.; Billeter, M.; Wüthrich, K. MOLMOL: A program for display and analysis of macromolecular
structures. J. Mol. Graph. 1996,14, 51–55.
55.
Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF
Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem.
2004
,25, 1605–1612.
Int. J. Mol. Sci. 2020,21, 9200 17 of 17
56.
Wu, D.; Chen, A.; Johnson, C.S. An improved diffusion-ordered spectroscopy experiment incorporating
bipolar-gradient pulses. J. Magn. Reson. Ser. A 1995,115, 260–264.
57.
Hwang, T.L.; Shaka, A. Water suppression that works. Excitation sculpting using arbitrary wave-forms and
pulsed-field gradients. J. Magn. Reson. Ser. A 1995,112, 275–279.
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... In the presented research, starting from the Phe-Arg-His (FRH) peptide motif (Scheme 1), which is known as an efficient ligand for Cu 2+ , and has a role in protecting synapses from copper-related oxidative damage by resisting ROS generation [30,31]; also belonging to the large family of short peptide sequences with a high affinity toward Cu 2+ [32], we wanted to address two goals. First, prepare fluorescent FRH-peptide analogs with triazole replacing histidine and study their interactions with Cu 2+ to see whether such analogs could replace FRH in biorelevant systems and allow easy and versatile "click" introduction of various fluorophores and other tools in the late-stage diversification of peptides/proteins. ...
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