The folding of a metallopeptide

Article abstract of DOI:10.1039/c5dt02797g

We have applied solid-phase synthesis methods for the construction of tris(bipyridyl) peptidic ligands that coordinate Fe(ii) ions with high affinity and fold into stable mononuclear metallopeptides. The main factors influencing the folding pathway and ch

Full text of DOI:10.1039/c5dt02797g

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RAMA MARTÍNEZ, E. Ortega, R. Berardozzi, V. M. Sánchez-Pedregal, L. Di Bari, J. Marechal, M. E. Vázquez
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PleaDsealdtoonnTortaandsjuascttimonasrgins
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DOI: 10.1039/C5DT02797G
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The folding of a metallopeptide
Ilaria Gamba,a Gustavo Rama,a Elisabeth Ortega-Carrasco,b Roberto Berardozzi,c Víctor M.
Sánchez-Pedregal,d Lorenzo Di Bari,c Jean-Didier Maréchal,*,b M. Eugenio Vázquez,*,d
Miguel Vázquez López*,a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We have applied solid-phase synthesis methods for the construction of tris(bipyridyl) peptidic ligands that coordinate Fe(II)
ions with high affinity and fold into stable mononuclear metallopeptides. The main factors influencing the folding pathway
and chiral control of the peptidic ligands around the metal ions has been studied both by experimental techniques (CD,
UV-vis and NMR) and molecular modeling tools. Amongst the numerous molecular variables that have been studied, this
study clearly illustrates how the chirality of a given set of aminoacids (proline in this case) of the peptide dictates the
chirality of the metal center of the resulting metallopeptide. Moreover, the reatively hydrophobic peptidic models used in
this work show that the most stable structures present reduced solvent contacts and, in couterpart, stabilize cis
configuration of the proline residues.
www.rsc.org/
difficult to establish with peptides coded by the 20 natural
amino acids, because too many physicochemical variables are
involved when the donor atoms suitable for coordination are
Introduction
Understanding the way in which peptides and proteins fold
into functional architectures in an autonomously-guided
mechanism defined by the amino acid sequence, that is, the
protein folding problem, has been a formidable scientific
located in the side chains of the peptide sequence. In contrast,
artificial metallopeptides, in which the metal-binding units are
part of the main chain of the amino acid structure, represent
excellent model systems for the study of metallopeptide
folding, as they can be described with far less variables and
offer much better coupling between the conformational
preferences of the peptide chain and the coordinating
properties of the metal ions.9,10,11 Herein, we present a
computational and experimental study of the folding and chiral
control of a family of octahedral mononuclear metallopeptides
containing metal-coordinating bipyridyl units as integral part
of the peptide backbone, and describe important factors that
influence the metal-directed folding of the peptide ligands into
chiral structures.
1,2
challenge since its identification more than 50 years ago.
Moreover, despite the increasing success in the prediction of
the three dimensional structures of small proteins and
peptides,3 the study of metallopeptides—how they fold and
misfold,4 aggregate,5 or interact with other molecules—is still
in its infancy.6,7 These studies are of great relevance, given the
role of metal ions in the folding/misfolding of metalloproteins
and also in relevant pathological processes, such as the
induction of amyloid aggregation and precipitation in
neurodegenerative diseases.8 One of the main reasons for this
underdevelopment is that the breakdown of the different
energies involved in the folding of metallopeptides is very
Results and discussion
2,2’-bipyridine (Bpy) is a privileged metal chelator and, as such,
has been extensively used in coordination and supramolecular
chemistry.12 We synthesized a Bpy analog appropriately
modified for its application in solid phase peptide synthesis
(SPPS) in which the Bpy unit was derivatized as a Fmoc-
protected amino acid with 5-amino-3-oxapentanoic acid
(Fmoc-O1PenBpy-OH, 1, Scheme 1). Following the synthesis of
the amino acid building block, we designed a set of six peptide
ligands featuring three metal-binding bipyridine units
connected by two short loops (LL-P, DD-P, GD-P, GL-P, DL-P and
LD-P, Scheme1). The loops include a β-turn promoting
sequence (Pro-Gly) that directs the folding of the peptide
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Journal Name
chains into discrete mononuclear species and encodes the
As expected, the enantiomeric LL-P and DD-PVileiwgaArntidclse Ognilvinee
chirality of their resulting complexes; the N-terminal loop rise to mirror image CD spectra characteDriOzeI:d10b.1y03t9w/Co5DbTa0n2d7s97oGf
may contain a chiral proline (–Gly-( )Pro-Gly– or –Gly-( )Pro- opposite sign at 300 and 329 nm with a crossover at 314 nm,
9
L
D
Gly–), or an achiral sequence (–Gly-Gly-Gly–), while the C- so that LL-P displays a positive couplet and DD-P a negative
terminal loop contains in all cases a chiral proline residue (– couplet. It is worth recalling that a CD couplet is defined a
Gly-(L)Pro-Gly– or –Gly-(D)Pro-Gly–). All the peptides were sequence of two bands of approximately equal amplitude and
obtained using standard Fmoc/tBu solid-phase protocols,13 and opposite signs, with crossover point close to the absorption
the final products were purified by reverse-phase HPLC and maximum; the couplet is defined positive/negative according
identified by MS (see ESI).
to the sign of its long-wavelength component. The intensity of
Following the synthesis of the peptide ligands, we first these bands increases upon addition of 1 equivalent of Fe(II)
studied the thermodynamic stability of their metal complexes. ions and their increase is also accompanied by bathochromic
Thus, incubation of low µM solutions of the peptide ligands shifts of both couplet components, respectively. Furthermore,
LL-P (featuring homochiral Pro loops), GL-P (one chiral loop), upon iron chelation we observe the appearance of another
and DL-P (heterochiral loops) at 298 K with Fe(II) ions resulted broad bisignate feature allied to a charge transfer transitions
in qualitatively similar changes, most notably
a clear around 550 nm. As observed in other cases before, this pair of
bathochromic shift of the Bpy absorption band from 308 to bands in the vis-region have opposite sign sequence with
approximately 316 nm due to the complexation processes (See respect to the UV-couplet.16 The sign of the UV-couplet is
ESI, Figures S1-S3). The binding constants derived from the consistent with a Λ configuration in the metal centre for [Fe(LL
UV/vis titrations indicate that all the complexes are very P)]2+ and with the opposite Δ configuration for [Fe(DD-P)]2+
stable, so that the values of the formation constants are β1,1
(Figure 1).17 Interestingly, the CD spectra of the LL-P, LD-P and
-
≈
8 for LL-P and GL-P, and β1,1 ≈ 9 for the more stable heterochiral DD-P systems indicate that a single Pro residue in the sequence
peptide complex with DL-P (ESI, Figures S4-6 and Table S1).14 (namely, the N-terminal Pro) encodes the dominating chirality
The assembly of the discrete mononuclear octahedral of the resulting metallopeptide. Thus, the CD spectra of LD-P
complexes was further confirmed by MALDI-TOF mass and LL-P, as well as those of their corresponding Fe(II)
spectrometry (See ESI).15
complexes, [Fe(LD-P)]2+ and [Fe(LL-P)]2+ display a positive UV-
couplet around 300 nm, and correspondingly
a
O
negative/positive sequence long-wavelength bands around
550 (Figure 1 and ESI, Figures S7-9). The presence of the UV
couplet before complexation and the conservation of its sign
(although with a significant increase in amplitude) after metal
binding suggests that the bipyridyl moieties in the free
peptides are to some extent preorganized to host the metal.
O
FmocHN
FmocHN
N
CO2H
H
N
N
Fmoc-O1PenBpy-OH (1)
SPPS, 12 cycles
TFA cleavage
N-terminus
H2N
N-loop
N-Pro
O
N-Gly2
H
O
N
N
N
H
N
N
C-loop
N
O
1.5
1
(D/L)
NH
C-Pro
O
N-Bpy
O
O
C-Gly2
NH
N-Gly1
(D/L)
X 10
O
O
N
HN
O
0.5
N
NH
O
C-Gly1
NH
O
O
0
-0.5
-1
M-Bpy
HN
O
H
N
N
N
NH2
C-terminus
O
-1.5
C-Bpy
O
250 300 350 400 450 500 550 600 650
(NH4)2FeSO4 • 6H2O
10 mM PBS
pH 5
λ / nm
Figure 1. a) CD spectra of [Fe(LL-P)]2+ (black line), [Fe(LD-P)]2+ (red line), and
[Fe(DD-P)]2+ (dashed line). CD spectra were measured at 293 K, 10 mM PBS
buffer, pH = 5.0.
+2
[Fe(P)]
N-loop
C-loop
–Gly–(L)Pro–Gly– –Gly–(L)Pro–Gly–
–Gly–(D)Pro–Gly– –Gly–(D)Pro–Gly–
–Gly–(L)Pro–Gly– –Gly–(D)Pro–Gly–
–Gly–(D)Pro–Gly– –Gly–(L)Pro–Gly–
LL-P:
DD-P:
LD-P:
DL-P:
GL-P:
GD-P:
While conducting these CD studies we became aware that
the assembly of the Fe(II) metallopeptides is a kinetically slow
process, and therefore we decided to monitor the folding of
the peptide ligands in presence of Fe(II) ions. The homochiral
ligands (LL-P and DD-P) displayed a monoexponential decrease
(or increase) of their 332 nm CD signal at 40 ºC upon addition
of Fe(II), reaching a plateau after approximately 25 min (Figure
2a), consistent with a two-state process in which the unfolded
–Gly–Gly–Gly–
–Gly–Gly–Gly–
–Gly–(L)Pro–Gly–
–Gly–(D)Pro–Gly–
Scheme 1. Solid phase peptide synthesis of the tris(bipyridyl) peptide ligands
LL-P DD-P LD-P DL-P GL-P and GD-P, as well as their corresponding Fe(II)
,
,
,
,
mononuclear octahedral complexes.
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peptides reach thermodynamic equilibrium as folded Fe(II)- heterochiral peptide ligand in the presence of Fe(II) ions shows
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metallopeptides (U ⇄ F). The simple two-state process is also four peaks (ESI, Figure S22), suggestingDtOhIe: 10p.r1e0s3e9/nCc5eDTo0f27fo97uGr
confirmed by the existence of a isodichroic point.18 In contrast major isomers in solution. Moreover, these four complexes are
with this, the intensity of the CD signal at 332 nm of the in equilibrium, as evidenced by the observation of the same
peptides containing two proline residues with opposite four peaks when each of the isolated peaks is injected back
chirality (DL-P and LD-P) displays a biphasic profile at 40 ºC, and into the HPLC after a short equilibration time. LL-P and GL-P
lacks the isodichroic point, so that a rapid increase (or Fe(II) metallopeptides also show the same behaviour.
1
decrease) in the CD intensity is followed by a slower Moreover, the solution H-NMR spectrum of [Fe(LL-P)]2+ shows
exponential decay (or increase) (Figure 2b). In this case, the a set of resonances compatible with four distinct isomers (ESI,
folding process cannot be simply described by the direct Figures S23-28). We tried to assess whether these isomers are
transition between an unfolded and a folded state (U ⇄ F), but in chemical exchange equilibrium by variable temperature
requires the consideration of an intermediate complex (U ⇄ I NMR in the 5-45 ºC range (ESI, Figure S29), but no sign of
⇄ F). Moreover, lowering the temperature from 40 ºC to 20 chemical exchange was evident in this range. This is
ºC resulted in an increase of the CD signal, but more compatible with a slow rate of interconversion between the
importantly, it also induced a change in the kinetic profiles of four isomers at the NMR timescale in the thermodynamic
the homochiral peptides, which become biphasic at 20 ºC, thus equilibrium suggested by the observations made by HPLC.
suggesting the accumulation at this temperature of an
Molecular modeling and QM/MM optimization provided
intermediate complex similar to that observed for the mixed 3D models of the possible Fe(II) isomers derived from the DL-P
chirality peptides also at higher temperatures. In contrast with ligand. These studies show that several (10) isomeric
this, the mixed chirality peptide, DL-P as well as those with a metallopeptide configurations differing in the folding of the
single Pro residue, GL-P and GD-P, maintain their folding peptidic chain around the metal center are energetically
profiles, albeit with at significantly slower rate (see ESI for the accessible (Figure 3 and ESI, Figures S30). It is worth noting
kinetic constants, Table S2).
that the lowest energy structure contain two cis prolines which
In all cases the observed changes in the CD only affect the suggests that during the metallopeptide assembly process
intensity of the bands, but not their position, and these these residues isomerize from their more stable trans
variations are not observed in the absorption spectra (ESI, conformation in solution20 to satisfy the conformational
Figures S13-21). This suggests that the variations in the CD requirements for metal coordination (see ESI Figure S31-33).21
intensity arise from changes in the relative position of the Bpy The energetic breakdown of the total QM/MM energy does
chromophores around the metal center as they rearrange to not show major constraints on the first coordination sphere of
their most stable geometry through non-dissociative the Fe(II) ion, and the fold of the rest of the peptide is mainly
mechanisms, such as the Bailar or the Ray–Dutt twists function of the energy of the loops (see ESI Figure S34).
isomerizations.19
However, when considering the complete set of physical
variables taken into account in our calculations, we observe
that the major factor influencing the relative stability of the
different isomers is the desolvation of the peptide chain that
favors the more compact structures.22
20 ºC
10 ºC
a
b
10 ºC
20 ºC
2
0
-0.5
-1
30 ºC
40 ºC
1.5
30 ºC
40 ºC
1
0
300 600 900 12001500
time (sec)
0
1800 3600 5400
time (sec)
Figure 2. Evolution of the circular dichroism signal at 332 nm of LL-P
(a), and DL-P (b) upon addition of Fe(II) at various temperatures. LL-P
Shows monoexponential decays at 40 and 30 ºC, and the biphasic
profiles only appear at lower temperatures. DL-P shows slower kinetics
(note the timescale of the plots) with biphasic profiles at all
temperatures.
Figure 3. Left, QM/MM relative energies of the possible Fe(II) complexes
formed with the DL-P peptide ligand. Δ isomers are shown in black and Λ in
light grey. The [Fe(DL-P)]2+ isomer labelled as Δ4, which has the lowest
energy of all the complexes, is represented on the right (bipyridines in
darker shade of grey). Please see Fig. S37 in order to compare this cis-Pro
isomer with a trans-Pro DL-P analogue.
At this point, we decided to further investigate the folding
process of the metallopeptide assembly by experimental and
theoretical tools. Surprisingly, HPLC analysis of the DL-P
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As we have seen, the CD spectra of the final mixtures of Figure 4. Left: Proposed complexation/folding mechVainewismArticloefOntlihnee
DOI: 10.1039/C5DT02797G
four isomers (Figure 1 and ESI, Figures S7-9) indicate an overall metallopeptides involving initial coordination to the peptide ligands
chiral selection governed by the chirality of the N-terminal Pro predominantly in trans-Pro configurations, and subsequent rearrangement
residue. Computation on the six metallopeptide systems (LL
-
and selection under thermodynamic control. Right: Circular dichroism of a
P/DD-P, GD-P/GL-P, DL-P/LD-P) are not entirely conclusive 110 µM solution of [Fe(LL-P)]2+ in 10 mM phosphate buffer pH 5.0 showing
regarding the fact that the overall chirality of the systems is the reversible modification of the CD signal (332 nm) in response to changes
independent of the chirality of the Pro residue in the C- in the temperature (30-40ºC).
terminal loop (ESI, Figures S31-33). Indeed, Δ4 and Λ4
structures are always close in energy in the six systems,
These theoretical predictions could be useful to explain the
despite presenting opposite chirality. We therefore look for a observations made during the CD vs time experiments above
possible dynamical reason of the N-terminal importance in described. In fact, the timescale of the kinetic profiles suggests
defining the chirality of the final species. Because the relative that the trans → cis isomerization of the proline residues is the
position of the Pro residues to the terminal bipyridine groups underlying process promoting the reorganization of the
could be responsible of a difference in their flexibility, we peptide ligands around the metal ions.23 This is in agreement
hypothesized that it could lead to major preorganization for with the known preference of Pro residues to present a trans
metal binding at the N-terminal end. An additional set of conformation in short peptides in solution,20 and the
calculations was therefore performed with a 1ns MD of DD-P molecular modelling studies showing that the M-Bpy/N-Bpy
and LL-P isolated ligands. The analysis of the MD shows that pair is significantly structured (and thus stabilizing an
the C-terminal Bpy unit (C-Bpy) is more flexible, and otherwise unstable cis proline in the N-loop). Moreover, Pro
conformationally less defined than the N-Bpy group (Scheme isomerization has also been proposed as the slow step in the
1), and also that the N-Bpy/M-Bpy pair is more preorganized denaturation/folding pathway of small proteins.24 The folding
than the M-Bpy/C-Bpy (ESI, Figure S35 and S36).
of these Fe(II) trisbipyridyl metallopeptides could be explained
The ECD spectra for the 10 structures discussed above for as follows (Figure 4, left). In the initial state the free peptide
the LD-P system (Figure 3; ESI, Figures S38-41) were simulated ligands are poorly structured (particularly their C-terminal
by means of time dependent DFT calculations (TD-DFT). To this end), and have their Pro residues in trans configuration; the
end, we pruned the chromophores by replacing the chains at addition of Fe(II) ions to the solution of the peptide ligands
the sides of the amide groups with H-atoms. All the hydrogen gives rise to a number of isomeric metallopeptides in
atoms were reoptimized with standard DFT (CAM- equilibrium, which progressively collapse into the observed set
B3LYP//SVP), clamping all the remaining atoms in their original of four isomers, which may have their Pro residues in the cis
positions. This truncation simplified the calculations, which conformation. Interestingly, the position of the equilibrium
otherwise would be excessively demanding, and it was between the selected isomers can be shifted by changes in the
justified by the fact that the chains connecting the bipyridyl temperature. Thus for example, varying the temperature of a
amides provide only weak spectroscopic contributions. TDDFT [Fe(LL-P)]2+ solution between 30 and 40 ºC results in
at the same level used for geometry optimizations (CAM- measurable changes in the CD spectrum, which can be
B3LYP/SVP) provided the ECD spectra for the individual reversibly switched between two extreme values (Figure 4,
conformations shown in Figures S39 and S40. The proximity right). Given the conformational manifold described above, the
and dissymmetric orientation of the three chromophores is reversibility displayed in this variable temperature experiment
responsible for the typical exciton couplet structure, with D is remarkable. Moreover, this figure reveals another important
and
L forms yielding positive and negative couplets, aspect: it describes the amplitude of a CD band due to exciton
respectively.
coupling of the bipyridyl groups. In a simple picture, where at
higher temperature corresponds looser structures, one would
expect that this amplitude should diminish, which is exactly
opposite to the experimental finding. This demonstrates that
between 30° and 40° C there occurs a relative population shift
between two stable conformations and not just a process of
melting of secondary structure.
1.3
1.2
1.1
1
40 ºC
Conclusions
In summary, we describe the folding process of a family of
trisbipyridyl Fe(II) mononuclear metallopeptides. The two-step
folding process involves a rapid coordination of the ligand
peptides to the Fe(II) ions and the formation of a number of
isomers (up to 10 possible species can be formed); this mixture
collapses into four well-defined isomers. Based on high-level
theoretical calculations and kinetic data, we suggest that the
key factor that drives the folding pathway is a trans → cis
30 ºC
0
120 240 360
time (min)
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isomerization of the Pro residues located in the loops of the
peptides connecting the three coordinating bipyridine units.
Moreover, we have also observed that the position of the
equilibrium between the four isomers can be reversibility
switched by varying the temperature of the solution mixture.
Finally, we have shown that there is an overall chiral selection
in the mixture of isomers in the equilibrium, and that it is
programmed by the chirality of the N-terminal Pro residue in
the peptide ligand. We believe that these studies could help to
improve the scarce knowledge about the folding mechanism in
metallopeptides and metalloproteins.
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DOI: 10.1039/C5DT02797G
11 I. Gamba, G. Rama, E. Ortega-Carrasco, J.-D. Maréchal, J.
Martínez-Costas, M. E. Vázquez and M. Vázquez López,
Chem. Commun., 2014, 50, 11097–11100.
12 C. Kaes, A. Katz and M. W. Hossein, Chem. Rev. 2000, 100,
3553.
13 I. Coin, M. Beyermann and M. Bienert, Nature Prot., 2007, 2,
3247.
14 The best fit to the experimental data suggests a significant
proportion of the 1:2 ML species in the initial steps of the
UV-vis titrations, when the peptidic ligand is in greater
excess over the metal ion. Further increase in the metal-to-
ligand ratios, progressively shifts the equilibrium towards the
expected 1:1 complex later in the titration.
15 The UV-vis spectra of the Fe(II) metallopeptides show
intense bands centered at 543 nm. These data, together with
the MALDI spectra, suggest octahedral coordination
geometries of the metal centers in their respective
complexes.
Acknowledgements
We are thankful for the support given by the Spanish grants
SAF2013-41943-R, CTQ2012-31341, CTQ2011-23336 and
CTQ2013-49317-EXP; the ERDF and the European Research
Council (Advanced Grant 340055); the Xunta de Galicia grants
GRC2013-041 and PGIDIT08CSA-047209PR and the Generalitat
de Catalunya grant 2009SGR68. Support of COST Action
CM1105 is kindly acknowledged. G.R. thanks the INL for his
PhD fellowship.
16 J. Dragna, G. Pescitelli, L. Tran, V. M. Lynch, E.V. Anslyn, L. Di
Bari, J. Am. Chem. Soc., 2012, 134, 4398-4407.
17 a) H. Mürner, P. Belser and A. Zelewsky, J. Am. Chem. Soc.,
1996, 118, 7989–7994; b) P. Wang, J. E. Miller, L. M. Henling,
C. L. Stern, N. L. Frank, A. L. Eckerman and T. J. Meade, Inorg.
Chem., 2007, 46, 9853–9862.
Notes and references
18 G. Pescitelli, L. Di Bari, N. Berova, Chem. Soc. Rev., 2013, 43,
5211-5233.
1
2
3
J. C. Kendrew, G. Bodo, H. M. Dintzis, R. G. Parrish and H.
Wyckoff, Nature, 1958, 181, 662–666.
19 A. Rodger and B. F. G. Johnson, Inorg. Chem., 1988, 27,
3061–3062; M. Amati and F. Lelj, Theor. Chem. Account,
2008, 120, 447–457.
20 a) J. Brandts, H. Halvorson and M. Brennan, Biochemistry,
1975, 14, 4953-4963; b) C. Dugave and L. Demange, Chem.
Rev., 2003, 103, 2475-2532.
21 J. F. Brandts, H. R. Halvorson and M. Brennan, Biochemistry,
1975, 14, 4953–4963.
22 O. Bignucolo, H. T. Leung, S. Grzesiek and S. Bernèche, J. Am.
Chem. Soc., 2015, 137, 4300–4303.
23 a) W. A. Thomas and M. K. Williams, J. Chem. Soc., Chem.
Commun., 1972, 994; b) D. Kern, M. Schutkowski and T.
Drakenberg, J. Am. Chem. Soc., 1997, 119, 8403–8408.
24 J. F. Brandts, H. R. Halvorson and M. Brennan, Biochemistry,
1975, 14, 4953–4963.
C. B. Anfisen, Science, 1973, 181, 223–230; b) K. A. Dill and J.
L. MacCallum, Science, 2012, 338, 1042–1046.
R. Schweitzer-Stenner, V. Uversky. Peptide Folding,
Misfolding & Nonfolding (Ed.: R. Schweitzer-Stenner). Wiley-
VCH. 2012.
D. Shortle, FASEB J., 1996, 10, 27–34.
A. Aguzzi and T. O'Connor, Nat. Rev. Drug. Discov., 2010, 9,
237–248.
4
5
6
V.
Lykourinou,
L.-J.
Ming
in
Metallofoldamers:
Supramolecular Architectures from Helicates to Biomimetics
(Eds.: G. Maayan, M. Albrecth), Wiley-VCH, 2013, pp. 1–50.
E. Ortega-Carrasco, A. Lledós, J.-D. Maréchal, J. R. Soc.
Interface, 2014, 11, 20140090
a) E. House, J. Collingwood, A. Khan, O. Korchazkina, G.
Berthon and C. Exley, J. Alzheimers Dis., 2004, 6, 291–201; b)
C. Exley, J. Alzheimers Dis., 2006, 10, 173–177; c) E.
7
8
Permyakov, Metallloproteomics, John Wiley
&
Sons,
Hoboken, New Jersey (USA), 2009.
9
G. Rama, A. Ardá, J.-D. Maréchal, I. Gamba, H. Ishida, J.
Jiménez-Barbero, M. E. Vázquez and M. Vázquez López,
Chem. Eur. J., 2012, 18, 7030–7035.
10 I. Gamba, I. Salvadó, G. Rama, M. Bertazzon, M. I. Sánchez, V.
M. Sánchez-Pedregal, J. Martínez-Costas, R. F. Brissos, P.
Gamez, J. L. Mascareñas, M. Vázquez López and M. E.
Vázquez, Chem. Eur. J., 2013, 19, 13369–13375.
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DOI: 10.1039/C5DT02797G
Graphical Abstract
The folding of a metallopeptide
Ilaria Gamba,¶ Gustavo Rama,¶ Elisabeth Ortega-Carrasco,# Roberto Berardozzi,† Lorenzo
Di Bari,† Víctor M. Sánchez-Pedregal,§ Jean-Didier Maréchal,#,* M. Eugenio Vázquez§,* and
Miguel Vázquez López¶,*
We have applied solid-phase synthesis methods for the construction of tris(bipyridyl)
peptidic ligands that coordinate Fe(II) ions with high affinity and fold into stable
mononuclear metallopeptides. The main factors influencing the folding pathway and
chiral control of the peptidic ligands around the metal ions has been studied both by
experimental techniques (CD, UV-vis and NMR) and molecular modeling tools
.