Transition-metal ion-mediated morphological transformation of pyridine-based peptide nanostructures

Article abstract of DOI:10.1039/d0nj04260a

Inspired by natural metallopeptides, we have rationally designed two pyridine-conjugated short peptides. These two peptide conjugates formed a pair of constitutional isomers that helped us describe their structure-function relationship. Both the isomers consisted of an equal number of aromatic amino acid residues, but shuffling was observed in the position of two key amino acids, viz; tyrosine and phenylalanine, which brought a remarkable change in their self-assembling behavior. The presence of specific functional groups and chemical diversity in both conjugates made them very active towards metal coordination. Both the constitutional isomers adopted different pathways of self-assembly, which could be further controlled or transformed by the use of transition metal ions. Interestingly, it was observed that the metal ions could precisely control the morphology of these metallopeptide nanostructures and make them more stable. Therefore, such artificial metallopeptides possess remarkable advantages over the natural counterparts primarily due to their tailor-made chemical structures.

Full text of DOI:10.1039/d0nj04260a

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Transition-metal ion-mediated morphological
transformation of pyridine-based peptide
nanostructures†
Cite this: New J. Chem., 2021,
45, 153
a
b
Narendra Singh,
Ramesh Singh,
Swati Sharma,a Khushboo Kesharwani,b
Khashti Ballabh Joshi *b and Sandeep Verma
*
a
Inspired by natural metallopeptides, we have rationally designed two pyridine-conjugated short peptides.
These two peptide conjugates formed a pair of constitutional isomers that helped us describe their
structure–function relationship. Both the isomers consisted of an equal number of aromatic amino acid
residues, but shuffling was observed in the position of two key amino acids, viz; tyrosine and phenyl-
alanine, which brought a remarkable change in their self-assembling behavior. The presence of specific
functional groups and chemical diversity in both conjugates made them very active towards metal
coordination. Both the constitutional isomers adopted different pathways of self-assembly, which could
be further controlled or transformed by the use of transition metal ions. Interestingly, it was observed
that the metal ions could precisely control the morphology of these metallopeptide nanostructures and
make them more stable. Therefore, such artificial metallopeptides possess remarkable advantages over
the natural counterparts primarily due to their tailor-made chemical structures.
Received 24th August 2020,
Accepted 10th November 2020
DOI: 10.1039/d0nj04260a
rsc.li/njc
Smartly designed short-peptide conjugates with appropriate flexible or rigid scaffold to a short peptide is one of the useful
functionality help us understand the structures and functions methods that provide thermodynamic and kinetic control
of several biologically relevant metal-binding proteins, which during the self-assembly process.16–18 By applying natural
may unlock ways to develop promising materials, devices, principles, several advances have already been made in the
and technologies of potential biotechnological interest.1–7 construction of nanostructures, including the self-assembly of
For biological functioning, approximately one-third of bio- nanofibrils, nanotubes, nanorods, nanorings, nanosheets,
molecules (proteins and enzymes) are activated by appropriate nanovesicles and helical ribbons, to name a few.20–26
co-factors i.e. metal ions5,6 because of their distinctive periodic
The formation of a specific secondary or three-dimensional
properties, including the presence of various coordinating structure primarily depends on the design of the basic skeleton
sites, electron-accepting ability, specific ligand affinity, positive of the molecular building blocks that self-assemble into well-
charge and different electronic structures.6–9 Short peptides defined and stable structures through covalent/non-covalent
equipped with a wide range of functional groups or metal bonds.20,25,27
chelating sites exhibit variable interactions with metal
The literature and our previous research work on synthesiz-
ions.1,2,8–12 Such metal-peptide composite biomaterials can ing meaningful nanostructures using peptides for various
mimic the functioning of natural metallopeptides, which possess applications28–32 demonstrate that the rational insertion of C2
various metal-binding sites, and therefore, are highly attractive symmetric flexible linkers, such as 1,2-diaminoethane,33 1,4-
systems for foreseeable future investigations.1,5,8,13,14
diaminobutane34 and 6-aminocaproic acid,35 has been exten-
Rationally designed short peptide conjugates along with small sively utilized to increase the ordering and transformation of
covalent linkers increase the ability to mimic the hierarchical non-fibrillating short peptides into fibrillating peptides.16,33–35
organization of natural proteins.8,15–17 The conjugation of a Further, the conjugation of a C3 symmetric scaffold, namely
tris(2-aminoethyl)amine (TREN), with the dipeptide Trp-Trp,
a Department of chemistry, Indian Institute of Technology, Kanpur-208016, India.
biotinylated ditryptophan peptide and others36–42 has been
exploited to form fascinating and highly precise nanocages or
rings,41 nanotorus37 and gold nanobangle36 structures. Other
than C2 symmetric flexible linkers, 2,6-pyridine dicarboxylic
acid and picolinic acid are known for their structural rigidity
and therefore, can control the symmetry of the conjugated
E-mail: sverma@iitk.ac.in
b Department of Chemistry, School of Chemical Science and Technology,
Dr HarisinghGour Central University, Sagar, MP, 470003, India.
E-mail: kbjoshi77@gmail.com
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj04260a
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peptide fragments.16,17 Such pre-defined rigidity in peptide characterization (ESI). The insertion of Tyr in the previously
fragments symmetrically appended on both sides can be used designed pyridine-diphenylalanine (Phe–Phe)19 conjugate
for numerous bio(nano)technological applications.17 There- increased the aromatic p–p interactions, H-bonding through
fore, in this milieu, this aromatic scaffold was conjugated to the sidechain and the ability to interact with specific metal ions
peptide conjugates of aromatic amino acids. In the first report, due to the presence of a phenolic group.
a ditryptophan peptide was linked and used to rescue clinically
These isomeric compounds 1 and 2 differ in the peptide
relevant bacterial cells from mercury toxicity.43 In another sequence only by the position of the tyrosine residue. Com-
report, the di-phenylalanine peptide was used to control coin- pound 1 has the tyrosine residue close to the pyridyl-scaffold,
age metal-mediated self-assembly modification and could be whereas compound 2 has the tyrosine residue far from the
used as a novel metallopeptide material to exploit interfacial scaffold i.e. at the terminal position (Scheme 1B bottom). Such
metal ion interactions.19
a molecular level change probably leads to a substantial con-
It can be predicted that a rigid linker minimizes the con- formational difference between these two peptides and hence
formational entropy changes, which results in the formation their self-assembly.
of highly organized supramolecular structures. It is well-known
We performed the structural analysis of both compounds by
1
that 2,6-pyridyl di-substituted type rigid linkers, such as 2,6- using spectroscopy techniques. H-NMR spectra were recorded
pyridine-dicarboxamide, have frequently been utilized to pro- for both the compounds (Fig. 1D). We observed that the
duce a helical conformation or a curvature in supramolecular aromatic and amide proton regions were the most sensitive;
structures.17,44–46
therefore, any degree of structural changes between 1 and 2 can
Therefore, inspired by the reported literature and based on easily be distinguished by the difference in the chemical shift of
our past research, we have rationally synthesized two isomeric these protons. Interestingly, it was observed that the signal for
compounds 1 and 2 (Scheme 1). The tripeptide conjugates the ortho protons of the Tyr side chain in peptide 1 was shifted
Tyr-Phe-Phe(N - C) and Phe-Phe-Tyr(N - C) were synthesized downfield compared to that of peptide 2 (Fig. 1D, blue box).
by solution-phase peptide synthesis47,48 (ESI) followed by con- The nature and splitting patterns of the Tyr and Phe aromatic
jugation to 2,6-pyridine dicarboxylic acid, forming conjugates 1 protons from 6.85 to 7.25 ppm (marked by blue arrows) were
and 2, respectively (Scheme 1). The resulting compounds found different for peptides 1 and 2, revealing that peptide 2
were purified by HPLC, followed by NMR and mass spectral existed in a more symmetric conformation compared with 1.
Further, the signal corresponding to the pyridyl proton (C4–H)
at 8.05 ppm was broader for 1 compared with that of 2 (Fig. 1D,
black circle),49,50 depicting the presence of an overcrowded
environment around the pyridine ring because the bulky Tyr
group is directly attached with the pyridine ring in 1. The signal
for other pyridyl protons (C3–H and C5–H) also showed a slight
Fig. 1 Top: FT-IR spectra of compounds 1 and 2. (A) Amide A and B
Scheme 1 (A) The representative synthesis scheme of the designed
isomeric short peptide conjugates 1 and 2; (B) structures of conjugates 1
and 2 and the corresponding predicted curvatures, which are supported by
(C) the reported molecule and its corresponding cartoon.19
regions ranging from 3600–3000 cmÀ1, representing N–H bond stretch-
ing; (B) amide I and II regions ranging from 1800 to 1500 cmÀ1; (C)
fingerprint region ranging from 1400 to 600 cmÀ1. Bottom: (D) The
1H-NMR spectra of compounds 1 and 2 in CD3OD.
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difference in chemical shift value (Fig. 1D, black box).49,50 The
conformational differences between the aromatic moieties in 1
and 2 were also confirmed by the Fourier transform Infrared
(FT-IR) spectral analysis. A clear difference in the out-of-plane
(Ar C–H) vibration band was observed (Fig. 1C) in the finger-
print region (1400 cmÀ1 to 600 cmÀ1). The FT-IR spectra B
for the free and H-bonded N–H vibrations in the amide
regions A and also showed clear differences in the nature of
H-bonding interactions (Fig. 1A). On the other hand, the amide
regions I and II, which disclose information about the second-
ary structure contribution and differences in the amide
backbone, showed very similar band positions at 1721, 1650
and 1521 cmÀ1 for 1 and 1720, 1647, 1519 cmÀ1 for 2 due to the
CQO carboxylic acid bond and amide I and II, respectively,
which support a similar kind of participation from both
molecules. Further deconvolution of the IR spectra in the
amide band I region revealed the quantitative analysis of the
secondary structural contribution in both the peptides (Fig. 2).
It was found that peptide 1 showed 100% contribution from the
a-helical conformation (Fig. 2B), while in peptide 2, it was
distributed between two structures, i.e. a-helix-64.45% and
b-sheet-34.55% (Fig. 2D).26,27
Fresh solutions of 1 and 2 (200 mM) in ethanol revealed the
formation of spherical self-assembled structures when deposited
over a freshly cleaved mica surface (Fig. 3). Noticeably, micro-
scopic (AFM and OM) observations revealed that these spherical
structures of 1 and 2 were different from each other in shape, size
and uniformity (Fig. 3). The SEM observations also corresponded
well with the AFM and OM observations.
The particle size distribution (PSD) histogram and subsequent
Gaussian fitting revealed that the self-assembled structures of
compound 1 were irregular and possessed diameters ranging
Fig. 3 (A) AFM image of the self-assembled structures of 1 in ethanol, (B)
the corresponding 3D image, and (C) the fluorescence optical image of the
fluorescein-stained sample of 1. (D) AFM image of the self-assembled
structures of 2, (E) the corresponding 3D image, and (F) the fluorescence
optical image of the fluorescein-stained sample. These images confirm
that 1 and 2 follow the different self-assembly processes.
from 50–350 nm (Fig. 4A, right) with an average value of
181.8 Æ 17.5 nm (Fig. 4A, right). However, the diameter of the
spherical structures of conjugate-2 ranged from 300–400 nm
with an average value of 345.8 Æ 2.0 nm (Fig. 3B, right). The
transmission electron microscopy (Fig. 5) and liquid cell AFM
imaging (ESI) observations also corresponded well with other
microscopy results and supported the formation of spherical
nanostructures with varied diameters. Therefore, it can be
envisaged that the obtained self-assembled nanostructures
are consistent in both liquid and air medium, showing
the negligible effect of the used hydrophilic and hydrophobic
surfaces, such as mica (AFM), carbon-coated copper grid (TEM),
copper surface (SEM) and glass surface (OM).
Fig. 2 (A) The FT-IR spectrum of 1 (solid line) and its deconvolution
(dashed line), and (B) the corresponding secondary structure contributions
to self-assembly. (C) The FT-IR spectrum of 2 (solid line) and its deconvo-
lution (dashed line), and (D) the corresponding secondary structure con-
tributions to self-assembly. Deconvolution was done by fitting multiple
Fig. 4 (A) Left: SEM images of the self-assembly of compound 1; Right:
The corresponding particle size distribution histogram of compound 1,
(B) Left: SEM images of the self-assembly of compound 2; Right: The
corresponding particle size distribution histogram of compound 2.
Gaussian peaks in the amide I region ranging from 1600 to 1700 cmÀ1
.
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Fig. 6 Fluorescence spectra of compounds 1 and 2 with the gradual
addition of metal ions. (A) Plot of the fluorescence intensity of compound 1
at 336 nm versus the concentration of added Fe3+ (K), Co2+
(
), Ni2+
(
),
); (B) plot of the fluorescence intensity of compound
2 at 336 nm versus the concentration of added Fe3+ (K), Co2+ ), Ni2+
), Cu2+ ) and Zn2+
). (C) Stern–Volmer plots of compound 1 with
Fe3+, Co2+, Ni2+, Cu2+ and Zn2+; (D) Stern–Volmer plots of compound 2
with Fe3+, Co2+, Ni2+, Cu2+ and Zn2+
Cu2+
( (
) and Zn2+
Fig. 5 TEM images of the self-assembled structures of (A and B) peptide 1
and (C and D) peptide 2.
(
(
(
(
.
Both tyrosine and phenylalanine are well-known to produce
ordered structures in solutions.51,52 Based on the literature,17,19 similar trends of quenching, whereas the addition of Ni2+
we also predicted that both 1 and 2 may engender a ‘crescent- showed fluorescence quenching to a lesser extent (Fig. 6B and
like’ rigid conformation with more metal ion co-ordination ESI†). Interestingly, opposite trends were observed when 2 was
sites for the interrogation of the peptide self-assembly titrated with Co2+ and Zn2+ ions, as these enhanced the
process.19 An appropriate position of tyrosine due to its p–p fluorescence intensity of 2 (Fig. 6B and ESI†). The corres-
and p–OH group interactions also provides a guiding effect27,53 ponding Stern–Volmer plots revealed that the order of com-
on the assembling structures in addition to the crescent-like plexation between 2 and the metal ions was Fe3+ Z Cu2+ 4 4
structure of the 2,6-pyridinedicarboxylic template,17,19,43,45 Ni2+ 4 Zn2+ 4 Co2+ (Fig. 6D). The effective quenching of the
thereby fostering a unique self-assembly pathway. Further, fluorescence intensity of both the peptides by the Fe(III) and
the intrinsic fluorescence of the tyrosine residue provides an Cu(II) ions can be explained by charge transfer.30,53 Very less or
additional advantage in monitoring the self-assembly process negligible quenching of both the peptides in the presence of
in the presence of external stimuli.27,30 The presence of phenyl- Ni(II) and Zn(II) ions is perhaps due to the condensed aggrega-
alanine along with tyrosine residues may lead to transient tion of the chromophores.27,56 Metal-ion enhanced fluores-
collocation in solution due to the p–p interactions and hydrogen cence (MEF) emission30,57 was also observed in the presence
bonds with adjacent residues and the amide backbone. Such of Co(II) for both 1 and 2, whereas changes in the fluorescence
interactions would reduce the lateral chain fluctuations and of peptide 2 in the presence of Zn(II) displayed an electric field
increase the incidences of metal ion coordination.
enhancement effect.30,57
Fluorescence titration measurements with different transi-
UV-Vis analysis was also done to support the obtained
tion metal ions were carried out (Fig. S9–S11, ESI†). Both 1 fluorescence results. A continuous increase in the absorbance
and 2 had the emission wavelength (lem) centred at 336 nm. intensity of 1 and 2 at 275 nm (lmax Tyr sidechain) was observed
The phenolic–OH group has a very good binding affinity for with the gradual addition of Fe(III) and Cu(II) solutions, which
Fe3+ ions.27,30,54,55 Therefore, a gradual decrease was observed revealed that almost all the added metal ions participated in
in the fluorescence intensity at 336 nm for both 1 and 2 on complexation (Fig. 7 and ESI†).
increasing the concentration of the Fe3+ ions (Fig. 6A and B and
Interestingly, the isomers showed different complexation
ESI†). In the case of the addition of Ni2+, Cu2+ and Zn2+ ions to behaviour with Fe(III) and Cu(II). Peptide 1 presented the order
1, quenching was observed to a lesser extent (Fig. 6A and ESI†), Fe(III) 4 Cu(II), while for peptide 2, it was reversed, i.e. Cu(II) 4
while Co2+ slightly increased the fluorescence intensity of 1 Fe(III) (Fig. 7C and D), which is probably due to the close
and 2. The Stern–Volmer plots for these quenching spectra location of the tyrosine sidechain to the terminal –COO group,
revealed that the order of complexation between 1 and the indicating that a certain amount of Cu(II) was bound to the
metal ions was Fe3+ c Cu2+ 4 Ni2+ 4 Zn2+ 4 Co2+ (Fig. 6C). In terminal –COO group. On the other hand, in the case of Co(II),
the case of compound-2, the addition of Fe3+ and Cu2+ followed Ni(II) and Zn (II), a negligible increase in the intensity at 275 nm
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Fig. 7 UV-Vis titration spectra of (A) 1 (40 mM) and (B) 2 (40 mM) with Fe(III)
as a representative; (C and D) increase in absorbance intensity of the
aromatic Tyr sidechain at 275 nm with the gradual addition of different
metal ions: Fe(III), Co(II), Ni(II), Cu(II), and Zn(II) to 1 and 2, respectively.
was observed for both the peptides. This early-stage saturation
of absorbance indicated that a small fraction of the Co(II), Ni(II)
and Zn (II) ions interacted with the Tyr residues. However, a
gradual increase in the absorbance intensity at 203 nm due to
the n - p* transition of the amidic and carboxylic –CO– groups
Fig. 8 SEM images of compound 1 in the presence of metal ions and the
indicated that all the metal ions were also coordinating with corresponding particle size distribution histograms, respectively, (A and B)
in the presence of Fe3+, (C and D) in the presence of Co2+, and (E and F) in
the amidic or carboxylic group. Further, from our previous
the presence of Ni2+
.
study19 and the fluorescence and UV data analysis, we could
predict that the Co(II), Ni(II) and Zn(II) ions were weakly inter-
acting with Tyr; however, they were preferably binding with the by scanning electron microscopy (SEM). Iron (Fe3+) has been
terminal carboxylic and amide groups.
previously reported to combine easily with the phenolic-OH
Transition-metal ions are well-known to play an important role in group of Tyr residues,27,30,54,55 and hence, we first investigated
the structural transformations of chelating biomolecules.10–12,30,36 the effect this metal ion had on the self-assembly of 1. The
Therefore, after examining the nature of the fluorescence of 1 addition of Fe3+ ions to 1 (1 : 1 composition) instantaneously
and 2 in the presence of these transition-metal ions and the increased the aggregation of 1 and minimized the diameter of
UV-Vis titration observations, we recorded the 1H-NMR spectra the spheres to the range of 40–80 nm (Fig. 8A). The corres-
of peptides 1 and 2 in the presence of Fe(III) ions-(as a ponding PSD analysis demonstrated that the diameter of these
representative) in order to know the positions of the peptide spherical structures significantly decreased, and the average
that predominantly coordinate with the metal ions (Fig. S19, diameter of the compound 1-Fe3+ hybrid structures was calcu-
1
ESI†). The H-NMR spectra of 1 and 2 in the presence of iron lated to be 68.3 Æ 0.9 nm (Fig. 8B). In the presence of Co2+ and
depicted that the nature of the aromatic-H signals was highly Ni2+ ions, interesting results were obtained. SEM observations
affected, along with a slight downfield shift. This perhaps is due revealed that the shape and size of the self-assembled spherical
to the aromatic side chain of the aromatic amino acids, including peptide-metal hybrids had a high degree of uniformity com-
pyridine, that mainly participate in metal-ion coordination. In pared with those made of the peptide alone (Fig. 8C and E).
addition to this, interestingly, the signal of the ortho protons of The particle size distribution histogram analysis demonstrated
the Tyr side chain in peptide 2 converted into a broad triplet-like that the size of these spherical structures had increased several
signal, while in the case of peptide 1, it remained a doublet but folds but with high precision and uniformity. The calculated
with some broadening. This difference in the NMR signals average diameters of the 1-Co2+ and 1-Ni2+ hybrid structures
between the peptides indicates the role of the aromatic sidechain were found to be 580.5 Æ 5.0 nm (Fig. 8D) and 466 Æ 3.4 nm
in the metal-ion interactions, with the tyrosine aromatic side- (Fig. 8F), respectively.
chain of peptide 2 showing significant binding with the metal
ions due to its feasible position.
On the other hand, in the presence of Cu2+ ions, a coales-
cence of soft structures was observed, and they subsequently
Then, the effect of these metal ions on the self-assembly self-arranged into an elongated chain-like assembly (Fig. 9A).
process of the sequence-shuffled isomeric peptides was examined This type of coalescence is perhaps due to coordination between
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Fig. 9 SEM images of compound 1 in the presence of metal ions and the
corresponding particle size distribution histogram, respectively, (A and B)
in the presence of Cu2+ and (C and D) in the presence of Zn2+ ions.
the peptide and the metal ions,19 followed by one-dimensional
growth (elongation into chains), the nucleation of which is
mediated via the liquid–liquid separation pathway.21 The
PSD analysis of the 1-Cu2+ hybrid structures revealed that the
average diameter of these spherical morphologies was 536.5 Æ
12.1 nm (Fig. 9B). Similarly, the presence of Zn2+ ions resulted
in the dimerization of the spheres (Fig. 9C), presenting an
average diameter of 481.8 Æ 8.7 nm (Fig. 9D). Likewise, the
effect of metal ions on the self-assembly of compound 2 was
also examined by using electron microscopy. We noticed
remarkable changes in the self-assembly patterns of these
hybrids. The addition of a Fe3+ solution into the solution of 2
in a 1 : 1 ratio composition led to minimized diameters of the
spheres by several folds (Fig. 10A). The particle size distribution
histogram revealed that the average diameter of the 2-Fe3+
hybrid structures was 74.5 Æ 5.0 nm (Fig. 10B). Unlike
other metal ions, Co2+ completely transformed the spherical
morphology into a circular multilayer (or stacked) sheet-like
morphology (Fig. 10C and D). Further, the addition of Ni2+ ions
also decreased the size of the spheres (Fig. 10E), and the
average diameter of the 2-Ni2+ hybrid structures was calculated
to be 53.3 Æ 6.0 nm (Fig. 10F).
Fig. 10 SEM images of compound 2 in the presence of metal ions and
the corresponding particle size distribution, respectively (A and B) in the
presence of Fe3+, (C and D) in the presence of Co2+, and (E and F) in the
presence of Ni2+
.
A slight or almost no change in the shape and size of spheres
of 2 was seen in the presence of Cu2+ ions (Fig. 11A). The
particle size distribution histogram revealed that the average
diameter of the hybrid structures of 2-Cu2+ was 406.7 Æ 6.3 nm
(Fig. 11B). On the other hand, in the presence of Zn2+ ions, the
spherical morphology was transformed into an aggregated
sheet-like morphology (Fig. 11C and D).
Fig. 11 SEM images of compound 2 in the presence of metal ions and the
corresponding particle size distribution, respectively, (A and B) in the
presence of Cu2+ and (C and D) In the presence of Zn2+ ions.
To understand the underlying mechanism of the metal-ion-
induced structural transformation, we recorded the FT-IR spectra
of both the peptides with and without metal ions. The Fourier The FT-IR spectra showed a strong band in the amide I region due
transform infrared spectroscopy (FT-IR) analysis validated the to the amide CQO stretching in all the tested samples of 1 and 2
structural transformation in the presence of Co2+/Zn2+ ions. (Fig. S16, ESI†). Deconvolution calculation was performed by
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fitting multiple peaks in the spectra ranging from 1600 to self-organization of 1 and 2.19,43 They also reveal that the
1700 cmÀ1. The deconvoluted spectra in the amide I region presence of tyrosine residue at its appropriate position perhaps
revealed that peptide 1 showed a very strong band centred at is the main guiding and controlling factor for achieving
1650 cmÀ1 (Fig. S16, panel ‘A’ green dotted line, ESI†) that self-assembly with different metal ions. The different and
could not be further disintegrated, which represented that the unique electronic features of these transition-metal ions cannot
main contribution was from a-helix-type secondary structures. be neglected. Since both the molecules are rich in aromatic
On the other hand, deconvolution of the amide I region of sidechains with properly positioned functionalities, aromatic
the peptide 2 spectrum showed disintegration into (Fig. S16, pi-interactions become additional predominant guiding fac-
panel ‘B’ red dotted line, ESI†) b-sheet-(1631 cmÀ1) and a-helix tors. The presence of amides and free carboxylic groups and
(1654 cmÀ1)-type secondary structures. The quantitative analysis the use of hydrophilic surfaces i.e. mica with lower contact
of the FT-IR spectra of peptides 1 and 2 revealed that the angles play a secondary role to further stabilize these struc-
obtained nano-assemblies of 1 had 100% a-helix contribution; tures. Further, the unique photo-physical property of the tyr-
however, for 2, 34.55% and 65.45% contribution from b-sheets osine residue provides an additional advantage in monitoring
and a-helices, respectively, was observed (Fig. S16 bar graph, the self-assembly process in the presence of external stimuli.
ESI†). Interestingly, the deconvolution analysis of the peptide 1 Therefore, based on the above experimental observations, the
spectrum in the presence of Co2+/Zn2+ ions showed negligible self-assembly processes of 1 and 2 with and without metal ions
effect (Fig. S16 panel A, ESI†) of these metal ions, and hence, the were predicted to a certain extent, as proposed in the model
morphology of the nanoassemblies was not affected much (Fig. (Fig. 12). Both the molecules may adopt a crescent-like shape,
8C, D and 9C, D). On the other hand, the deconvolution analysis which subsequently self-assembles into a vesicular construct.
of the peptide 2 spectrum in the presence of Co2+/Zn2+ ions The position of tyrosine in 1 destabilizes the proper aromatic
showed an entirely different nature of peaks with a complete interaction engendered between/by the pyridyl ring and FF
transition of the a-helix/b-sheet-type secondary structures residues; therefore, irregular shapes and sizes are initially
into antiparallel b-sheet/random coil-like secondary structures, obtained for the vesicular assemblies. However, the addition
which probably is the reason for the Co2+/Zn2+-induced struc- of appropriate transition metal ions help stabilize the electronic
tural transformation that results in a sheet-like morphology contribution of the phenolic –OH of tyrosine; therefore, the
(Fig. 11C, D and 10C, D).
aromatic interaction between the pyridyl and FF residues is in
Thus, based on these results, the following possibilities proper agreement, leading to a uniform spherical morphology
could be envisaged for metallopeptides 1 and 2. Microscopic and also a substantial increase in the vesicle diameter. Since
and spectroscopic evidence reveals that the crescent-shape Fe3+ preferably and strongly coordinates with Tyr–OH,27,30 a very
of the monomer engendered by the pyridyl linker helps in the high degree of coalescence along with a decrease in vesicle size
Fig. 12 Proposed model of the metal-peptide interactions and the different self-assembly processes followed by conjugates 1 and 2 alone and in the
presence of transition-metal ions.
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was observed. However, in the presence of Cu2+ ions, beautifully high degree of regularity were obtained with cobalt, nickel and
arranged coalesced vesicles were visible. zinc. The second isomer, wherein, the Tyr residues were at
On the other hand, in conjugate 2, the position of the the terminal of the crescent structure produced more refined
tyrosine residue is fixed at the terminal position of the crescent. and regular nanostructures compared with 1 due to the
Therefore, perhaps the presence of tyrosine at this position is aromatic homogeneity/symmetry in the structure. However,
more appropriate and stabilizes the aromatic pi-interactions. this homogeneity was perturbed in the presence of metal ions,
The phenolic –OH group may participate in intermolecular or and the vesicular morphology transformed into stacked and
intramolecular hydrogen bond formation with the carboxyl/ aggregated sheet-like morphologies in the presence of cobalt
phenolic –OH groups of its counterpart residues, thereby lead- and zinc, respectively. In the presence of nickel and iron, the
ing to the formation of structures with high precision. However, large vesicular morphology disintegrated, and with copper, a
in the presence of metal ions, the end tyrosine residue pre- smaller degree of coalescence was observed. Thus, it can be
ferably coordinates with the metal ions and hence tries to summarized that the shuffling of one or more key amino acids
transform the morphology too. In the presence of Fe3+ and in rationally designed peptide conjugates can be of great
Ni2+, the larger vesicles disintegrate into smaller ones; however, significance.
in the presence of Cu2+, vesicles of almost the same size were
obtained with some degree of coalescence. However, stacked
Conflicts of interest
and aggregated sheet-like morphologies were obtained in the
presence of Co2+ and Zn2+, respectively (Fig. S16, ESI†).
There are no conflicts to declare.
These self-assembled nanostructures of the metallopeptides
are governed by the functional properties of the peptides and
metal-peptide interactions. Therefore, the coordination of Acknowledgements
specific metal ions further provides novel metallopeptides of
NS, RS and SS thank to CSIR-UGC, UGC and MHRD for pre-
choice, which can be considered as bioactive metal ion reser-
doctoral research fellowship. KK thanks to DHSGSU for doc-
voirs for potential biotechnological applications. For example,
toral fellowship. NS and RS contributed equally to this work. SV
the order of the binding abilities may be utilized for the
would like to thank the J. C. Bose Fellowship (SERB, India) for
capture, displacement and delivery of clinically relevant metal
financial support. This work is partially supported by DST Nano
ions. This study on metallopeptides may be useful for enhan-
Mission and CSIR. We acknowledge Sophisticated Instrument
cing the ability of drug delivery systems through the drug
Centre (SIC) Dr Harisingh Gour Central University Sagar, M.P.,
displacement strategy.7,58 Because of their metal-binding
India for AFM. Our acknowledgement is incomplete without
strength, these peptide structures can also be utilized to disrupt
thanking Jai Kumar, IIT-Kanpur, for TEM.
the cell walls of metal ion-containing pathogens, which would
result in subsequent cell lysis.59 Such metallopeptide-based
nanostructures can also be utilized for bio-mimicking numerous
Notes and references
metalloenzymes involved in catalysis.60,61
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