Photophysical study of a π-stacked β-sheet nanofibril forming peptide bolaamphiphile hydrogel

Article abstract of DOI:10.1039/c3nj00814b

We describe the state of molecular self-assembly of a peptide based bolaamphiphile molecule using spectroscopic and microscopic techniques. The tryptophan and phenylalanine containing peptide bolaamphiphile forms a hydrogel upon sonication under physiological conditions. Sonication helps to reorient the peptide molecules by providing the required energy for the self-assembly process. The disassembly and self-assembly processes are influenced by various stimuli, including heating-cooling and shaking-rest methods. The extensive hydrogen bonding and π-π stacking interactions are responsible for the self-assembly process, which is confirmed by FT-IR, temperature dependent NMR and fluorescence spectroscopy studies. FT-IR and powder X-ray diffraction studies reveal that the gelator molecules self-assemble into an antiparallel β-sheet type structure. The TEM image of the hydrogel shows a well-defined amyloid-like nanofibrillar structure. The amyloid-like behaviour of the fibril forming peptide bolaamphiphile hydrogel is confirmed by ThT and Congo red binding studies. The effect of concentration, time and temperature on the self-assembly mechanism of the peptide bolaamphiphile hydrogel is investigated by time resolved fluorescence spectroscopy. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014.

Full text of DOI:10.1039/c3nj00814b

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Photophysical study of a p-stacked b-sheet
nanofibril forming peptide bolaamphiphile
hydrogel†
Cite this: New J. Chem., 2014,
38, 376
Indrajit Maity, Tushar K. Mukherjee* and Apurba K. Das*
We describe the state of molecular self-assembly of a peptide based bolaamphiphile molecule using
spectroscopic and microscopic techniques. The tryptophan and phenylalanine containing peptide
bolaamphiphile forms a hydrogel upon sonication under physiological conditions. Sonication helps to
reorient the peptide molecules by providing the required energy for the self-assembly process. The
disassembly and self-assembly processes are influenced by various stimuli, including heating–cooling
and shaking–rest methods. The extensive hydrogen bonding and p–p stacking interactions are responsi-
ble for the self-assembly process, which is confirmed by FT-IR, temperature dependent NMR and
fluorescence spectroscopy studies. FT-IR and powder X-ray diffraction studies reveal that the gelator
molecules self-assemble into an antiparallel b-sheet type structure. The TEM image of the hydrogel
shows a well-defined amyloid-like nanofibrillar structure. The amyloid-like behaviour of the fibril forming
peptide bolaamphiphile hydrogel is confirmed by ThT and Congo red binding studies. The effect of
concentration, time and temperature on the self-assembly mechanism of the peptide bolaamphiphile
hydrogel is investigated by time resolved fluorescence spectroscopy.
Received (in Montpellier, France)
23rd July 2013,
Accepted 16th October 2013
DOI: 10.1039/c3nj00814b
www.rsc.org/njc
and Parkinson’s disease.34–36 The peptides and proteins aggregate
into a b-sheet structure, which is common to all amyloid fibrils.
1. Introduction
A wide range of scientific investigations have established a growing Another example of amyloid disease is type II diabetes, where
interest in peptide-based hydrogels,1–4 which form through the misfolded islet amyloid polypeptides (IAPP) form amyloid fibrils in
self-assembly5–10 of small peptide molecules. In many cases, it is the pancreas.37 Amyloid fibrils are large, highly ordered polypeptide
necessary to use external stimuli such as enzymes,11–14 light,15,16 aggregates with high mechanical and chemical stability, which
heat,17,18 sonication19,20 or change in pH,21,22 which have a drastic have several applications in nanotechnology.38–44
effect on the self-assembly mechanism. These supramolecular
Bolaamphiphiles are another interesting class of amphi-
nanostructured hydrogels have been used in drug delivery philic molecules, which can self-assemble to form well-defined
systems,23–25 tissue engineering26–29 and various fields of nano- nanostructures. Generally, two hydrophilic end groups are
technology.30–32 The self-assembly of molecules starts from a covalently bonded with a hydrophobic spacer in a bolaamphi-
nucleation point and finally ends with higher ordered supra- phile molecule. Recently, several groups have explored the self-
molecular nanostructured materials. This process is dynamic assembly process of bolaamphiphiles.45–48 Amino acid and
in nature,33 strongly depends on the surrounding environment sugar based bolaamphiphiles have also been reported, which
and is dominated by non-covalent interactions. Therefore, it is can be used for gene delivery.49 Matsui and co-workers have
essential to investigate the detailed mechanism of the self- reported peptide bolaamphiphiles which self-assemble to form
assembly process to understand the fundamental non-covalent nanotubes.50 L-Glutamic acid-based bolaamphiphiles also self-
interactions at the supramolecular level.
assemble and form a hollow nanotubular structure.51,52
Currently, we are focused on stimuli responsive peptide
Peptide or protein mis-folding and anomalous aggregation into
amyloid fibrils are responsible for several neurodegenerative dis- bolaamphiphile self-assembly, which can tune the nanostructure
eases like Alzheimer’s disease, Prion disease, Huntington’s disease at a supramolecular level. We have designed peptide bolaamphi-
phile molecules with two different hydrophobic dipeptides,
which are covalently connected by a succinic acid moiety. The
balance between the hydrophobic and hydrophilic groups can
Department of Chemistry, Indian Institute of Technology Indore, Indore, India.
E-mail: apurba.das@iiti.ac.in; Fax: +91-731-2364-182
lead the peptide bolaamphiphile to self-assemble in aqueous
media. There are hydrogen-bonding sites in the two dipeptides,
† Electronic supplementary information (ESI) available: fluorescence decay and
synthesis of peptide bolaamphiphiles. See DOI: 10.1039/c3nj00814b
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along with two carboxylic acid groups. The hydrophobic aromatic (HO-Trp-Phe-Suc-Leu-Trp-OH) is capable of forming a transparent
side chains can also take part in the self-assembly process hydrogel in phosphate buffer (pH 8, 10 mmol) under physiological
through p–p stacking interactions. These molecules are ideal conditions (Fig. 1B), but the peptide bolaamphiphile 2 (HO-Phe-
for designing self-assembling soft materials through extensive Leu-Suc-Leu-Phe-OH) is unable to form a gel under the same
hydrogen bonding and p–p stacking interactions.
conditions. The self-assembly process of peptide bolaamphiphile 1
The self-assembly process of peptides and proteins has been was studied using various spectroscopic and microscopic techni-
studied using infra-red spectroscopy, fluorescence, powder ques along with time-resolved fluorescence spectroscopy.
X-ray diffraction, rheological studies and various microscopic
2.2. Effect of stimuli
techniques. The secondary structure of peptides and proteins
is analyzed by CD and FT-IR spectroscopy.53–55 Fluorescence The self-assembly behaviour of the peptide bolaamphiphile hydro-
spectroscopy provides direct information about p–p stacking gel 1 is influenced by external stimuli. The peptide bolaamphiphile
interactions, which play a role during the self-assembly process 1 was dispersed in phosphate buffer and sonicated for 30 minutes
of peptides/proteins containing aromatic residues.56,57 MacPhee to form a self-supporting hydrogel. Sonication helps to dissolve
et al. reported peptide self-assembly using a time correlated the peptide bolaamphiphile molecules in aqueous solution and
single photon counting (TCSPC) technique. In this case, the provides sufficient energy to reorient the molecules into a self-
peptide is modified with a fluorophore group (Fmoc), which assembled soft material through extensive hydrogen bonding
is capped with an N-terminus.58 Early aggregation in the self- and p–p stacking interactions.60 The peptide bolaamphiphile
assembly of prion peptides is characterized by nonlinear and 1 forms a thermoreversible and mechano-sensitive hydrogel
ultrafast time-resolved fluorescence spectroscopy.59 As a con- (Fig. 2A). The hydrogel turns to solution on heating, and turns
sequence, the study of the self-assembly process of a peptide back into a gel again at room temperature. The gel turns to
bolaamphiphile hydrogel by time-resolved fluorescence spectro- solution on shaking, but it turns back into a gel on rest. Thus,
scopy is rare. Our objective is to use time-resolved fluorescence heating and mechanical shaking disassemble the peptide
spectroscopy and other spectroscopic and microscopic techniques bolaamphiphile molecules by rupturing the non-covalent inter-
to establish the self-assembly process of a peptide bolaamphiphile actions. The peptide molecules again self-assemble and regain
based hydrogel.
their gelation property on cooling and resting (Fig. 2B).61
In the present study, we have used a tryptophan and phenyl-
alanine rich peptide bolaamphiphile 1 which self-assembles into
a b-sheet structure and forms a self-supporting hydrogel. The
hydrogel was characterized by FT-IR, temperature dependent
NMR, TEM, powder X-ray diffraction and rheological experi-
ments. The photophysical behavior of the self-assembled peptide
was explored by means of steady state and time resolved fluores-
cence spectroscopy.
2.3. Rheological and FT-IR studies
The dynamic mechanical behaviour of hydrogel 1 was investi-
gated by a rheological study. Fig. 3A shows that the storage
modulus (G0) exceeds that of the loss modulus (G00) over the
oscillating frequency. In the non-destructive frequency sweep
mode, the storage modulus dominates the loss modulus, exhibit-
ing the characteristics of a typical solid-like material.62,63 The gel is
moderately stiff, with G0 > 105 Pa at low frequency. The value of the
storage modulus G0 exceeds that of the loss modulus G00 by a
factor of 8 in the higher frequency region, which indicates the
2. Results and discussion
2.1. Hydrogel formation
Two peptide bolaamphiphiles containing a centrally located succinic
acid moiety attached to dipeptides have been synthesized for
the self-assembly study (Fig. 1A). The hydrogelation behaviour of
both peptide bolaamphiphiles has been investigated upon soni-
cation under physiological conditions. Peptide bolaamphiphile 1
Fig. 1 (A) Structures of peptide bolaamphiphiles 1–2 and (B) a photograph
of the self-supporting hydrogel formed from peptide bolaamphiphile 1.
Fig. 2 (A) Stimuli responsive assembly–disassembly of peptide bolaamphi-
phile 1 and (B) a scheme of the assembly–disassembly of 1.
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formation of a strong and rigid hydrogel. The FT-IR study of the 2.4. Steady state fluorescence study
solid compound and its corresponding hydrogel was performed
Peptide bolaamphiphile hydrogel 1 shows an absorption band
at 280 nm (Fig. 4A (i)). Excitation at 280 nm results a in clear
emission band centered at 355 nm (Fig. 4A (ii)). The emission
spectrum is quite broad and shows a weak shoulder at around
460 nm. Fig. 4B shows the effect of peptide bolaamphiphile
concentration on the fluorescence behaviour. The fluorescence
intensity increases with the increase in peptide bolaamphiphile
concentration from 2 mmol Lꢀ1 (solution state) to 10 mmol Lꢀ1
(gel state) with no significant peak shift. However, further increase
in peptide bolaamphiphile concentration to 25 mmol Lꢀ1 results
in a decrease in fluorescence intensity with a peak shift of B9 nm.
Interestingly, the weak shoulder at 460 nm is observed only at and
above 10 mmol Lꢀ1 concentration. To determine the origin of this
weak shoulder at 460 nm, we recorded the excitation spectrum65
with the emission wavelength fixed at 460 nm. The excitation
spectra of the peptide bolaamphiphile hydrogel recorded at
emission wavelengths of 355 nm and 460 nm differ signifi-
cantly (Fig. 5A). The excitation spectrum for the 355 nm emis-
sion band shows a peak at 290 nm with a shoulder at 266 nm,
which is similar to that of the absorption spectrum with a
10 nm red shift. On the other hand, the excitation spectrum for
460 nm shows a maximum peak at 296 nm and a new red
shifted broad band at around 365 nm. Excitation at 365 nm
results a large Stokes shifted emission band centered at 450 nm
at a concentration of 10 mmol Lꢀ1 (Fig. 5B (ii)). The fluorescence
intensity of this band is strongly dependent on the peptide
bolaamphiphile concentration (Fig. 5B). The emission at 450 nm
was observed for peptide bolaamphiphile 1 at and above
10 mmol Lꢀ1 concentration. No such emission was observed
for peptide bolaamphiphiles in solution phase at a concen-
tration of 2 mmol Lꢀ1 (Fig. 5B). Interestingly, the fluorescence
intensity at 450 nm increases with the increase in peptide
bolaamphiphile concentration up to 20 mmol Lꢀ1. Subsequent
increase in concentration to 25 mmol Lꢀ1 results in a decrease in
to investigate the secondary structural arrangement of the pep-
tide bolaamphiphile hydrogel (Fig. 3B). A N–H band at around
3298 cmꢀ1 was observed for solid compound 1, which indicates
hydrogen bonding interactions in the solid state. In the gel state,
the peak was shifted to 3040 cmꢀ1, which indicates a greater
extent of hydrogen bonding interactions. The peak at 2939 cmꢀ1
is ascribed to the CH3 stretching band of the leucine moiety in
the gel state. The peak of the carboxylic acid group appeared at
1718 cmꢀ1 in the solid state, and shifted to 1710 cmꢀ1 in the gel
state. The shift of the corresponding acid peak suggests that a
large amount of hydrogen bonded carboxylic acid groups are
involved in the hydrogelation process. The CQO stretching
band at 1736 cmꢀ1 can be assigned to non-hydrogen bonded
carboxylic acid groups.64
The amide I band at 1636 cmꢀ1 along with a peak at 1615 cmꢀ1
indicates the formation of a hydrogen bonded supramolecular
b-sheet structure in the gel state. Another band at 1538 cmꢀ1 is
assigned to the amide II band, which is another characteristic
feature of the b-sheet arrangement. Furthermore, a weak inten-
sity peak at 1680 cmꢀ1 suggests that the peptides are arranged in
an antiparallel manner to form a higher order supramolecular
b-sheet structure.55
Fig. 3 (A) Dynamic rheology (frequency sweep) of the hydrogel of pep-
tide bolaamphiphile 1 (C = 10 mmol Lꢀ1). The storage modulus G0 is higher
than the loss modulus G00. G0 > 105 Pa at low frequency, and the storage
modulus is higher than the loss modulus by a factor of 6–10, indicating the
excellent solid-like properties of the gel materials. (B) FT-IR spectrum
of the hydrogel of peptide bolaamphiphile 1, indicating an anti-parallel
b-sheet structure in the gel phase.
Fig. 4 (A) Absorption and emission spectra of peptide bolaamphiphile
hydrogel 1 at 10 mmol Lꢀ1 (lex = 280 nm). (B) Emission spectra of peptide
bolaamphiphile 1 at different concentrations: (i) 2 mmol Lꢀ1, (ii) 10 mmol Lꢀ1
and (iii) 25 mmol Lꢀ1
.
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the species responsible for the characteristic emission is only
formed with the formation of the gel state. Earlier, MacPhee
and coworkers have observed a similar kind of large Stokes
shifted emission (B440 nm) along with the normal emission
(B302 nm) from nanoscale fibrillar aggregates.58 Based on their
results they assigned the red shifted emission peak to excimer
formation. However, our results clearly signify that peptide
bolaamphiphiles in the gel state form higher order aggregates in
the ground state through p-stacking interactions between aromatic
moieties.69 These aggregates absorb near 365 nm and show
characteristic emission around 450 nm. At higher concentrations
these aggregates are self-quenched due to close proximity of the
peptide bolaamphiphiles. To further establish this proposed
model, we performed excited state decay measurements.
2.5. Time resolved self-assembly study
With our current time correlated single photon counting (TCSPC)
setup, we measured decay traces of the peptide bolaamphiphiles
with an excitation at 375 nm (Fig. 6A). Here, it is important to
remember that only the aggregated peptide bolaamphiphiles
show characteristic emission at 460 nm with 375 nm excitation.
All the traces have been fitted with a three exponential function.
The average lifetime of peptide bolaamphiphiles in the liquid
state at 2 mmol Lꢀ1 concentration is 0.32 ns, with lifetime com-
ponents of 0.68 ns (16%), 1.05 ns (80%) and 3.15 ns (0.04%).
However, in the gel state at a concentration of 10 mmol Lꢀ1, the
average lifetime increases to 0.94 ns with lifetime components
of 0.24 ns (62%), 1.19 ns (28%) and 4.76 ns (10%), which is very
similar to the increases in fluorescence intensity observed in the
steady state experiment. Further increase in peptide bolaamphi-
phile concentration up to 20 mmol Lꢀ1 results in a gradual
increase in lifetime. However, at 25 mmol Lꢀ1 concentration the
Fig. 5 (A) Excitation spectra of peptide bolaamphiphile hydrogel 1 at
350 nm (dashed line) and 450 nm (dotted line) overlapped with the absorp-
tion spectrum (solid line). (B) Fluorescence spectra of peptide bolaamphi-
phile 1 in solution (i) and as a hydrogel at (ii) 10 mmol Lꢀ1, (iii) 20 mmol Lꢀ1
and (iv) 25 mmol Lꢀ1 concentration with 365 nm excitation.
fluorescence intensity. This decrease in fluorescence intensity is
accompanied by a 9 nm red shift in the fluorescence spectrum.
The initial increase and subsequent decrease in fluores-
cence intensity at 355 nm signifies the formation of some kind
of stable ordered arrangement of peptide bolaamphiphiles
below 25 mmol Lꢀ1
.
At 2 mmol Lꢀ1 concentration, peptide bolaamphiphiles in the
solution state show very low fluorescence due to non-radiative
decay processes, mainly intramolecular torsional motion of the
aromatic rings. In the gel state at 10 mmol Lꢀ1 concentration, all
non-radiative torsional motion of the aromatic rings freezes,
resulting in enhanced fluorescence. The observed fluorescence
quenching at 25 mmol Lꢀ1 concentration may be due to either
destabilization of the ordered arrangement, or self-quenching
(concentration quenching) due to the very close proximity of
peptide bolaamphiphiles. However, the peptide bolaamphiphile
remains in the gel form even at 25 mmol Lꢀ1 concentration and
shows a 9 nm red shift in the fluorescence spectrum. This
indicates that at and above 25 mmol Lꢀ1 concentration the
peptide bolaamphiphiles are very closely packed, which leads
to self-quenching due to non-radiative energy loss.66,67 The
different excitation spectra at two different emission wave-
lengths (365 nm and 450 nm) clearly signifies that the mole-
cular origin of the two emission bands is different. This kind of
different excitation spectra at different wavelengths often signifies
ground state heterogeneity.68 The absence of a similar band in
Fig. 6 (A) Decay traces of peptide bolaamphiphile 1 at (ii) 2 mmol Lꢀ1
,
(iii) 10 mmol Lꢀ1, and (iv) 25 mmol Lꢀ1. (B) Decay traces of peptide
bolaamphiphile 1 hydrogel (10 mmol Lꢀ1) at (ii) 8 h, (iii) 1 day and (iv) 4 days.
the solution phase at 2 mmol Lꢀ1 concentration indicates that Curve (i) represents the IRF.
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average lifetime decreased to 0.75 ns with lifetime components of hydrogel at a concentration of 10 mmol Lꢀ1 was varied from
0.08 ns (66%), 0.96 ns (20%) and 3.60 ns (14%) (ESI,† Table S1). 15 1C to 80 1C. At 15 1C, the hydrogel shows an average lifetime
The very short lifetime at 2 mmol Lꢀ1 concentration (solution of 1.30 ns, which gradually decreases upon increasing the
state) of the peptide bolaamphiphiles indicates the involvement temperature (Fig. 7B), and at 80 1C it shows a value of 0.31 ns.
of non-radiative decay processes, mainly intramolecular torsional Interestingly, the average lifetime at 80 1C matches exactly with
motion of the aromatic rings of free peptide bolaamphiphiles. that of the solution state of the peptide bolaamphiphiles with
However, in the gel state, the molecules self-assemble to form a 2 mmol Lꢀ1 concentration. These results (ESI,† Table S3) strongly
rigid framework of b-sheet structures through p-stacking and suggest that the aggregated peptide bolaamphiphile slowly dis-
hydrogen bonding interactions, which results in increased life- sociates into monomeric units with the increase in temperature.
time.70,71 In this b-sheet structure, the intramolecular torsional At 80 1C, which is just above the gel melting temperature of the
motion of aromatic rings is hindered, and the excited state non- hydrogel, a phase transition occurs and all the peptide bolaamphi-
radiative decay process is somewhat hampered. Further increase phile molecules disassembled into the monomeric units.
in lifetime with increasing peptide bolaamphiphile concentration
1
2.6. Temperature dependent H NMR study
indicates a more compact ordered arrangement, in which the
aggregates are further stabilized. However, at 25 mmol Lꢀ1
1
The H NMR technique was used to confirm the p–p stacking
concentration the lifetime decreases, similar to that observed in
our steady state fluorescence data. Interestingly, at 25 mmol Lꢀ1
concentration, a new lifetime component with a very short decay
(0.08 ns) time has been observed. This major (66%) decay
component having a lifetime of 0.08 ns was neither observed
in the solution state nor in the gel states with lower concen-
tration. These results clearly support the idea that at and above
25 mmol Lꢀ1 concentration, peptide bolaamphiphiles are packed
very close to each other which results in self-quenching due to
non-radiative energy loss.72 Fig. 6B shows the decay traces of the
peptide bolaamphiphiles at a concentration of 10 mmol Lꢀ1 with
varying time at room temperature. The average lifetime of the gel
after 8 h is 0.87 ns, while that after 4 days is 1.12 ns. The change
in lifetime is very minor but noticeable. This nominal increase
in lifetime (ESI,† Table S2) with time indicates the progressive
arrangement of peptide bolaamphiphiles into the ordered
b-sheet structure. The effect of temperature on the gelation
dynamics has been investigated by recording their decay traces
at different temperatures (Fig. 7A). The temperature of the
interactions between the aromatic residues of peptide bola-
amphiphile 1 during the self-assembly process. The hydrogel
(10 mmol Lꢀ1) was prepared in sodium phosphate buffer in
D2O, and the temperature dependent 1H NMR study was
performed (Fig. 8). The study mainly focused on the aromatic
region of the peptide bolaamphiphile molecule to confirm the
p–p stacking interactions. In the gel state, the NMR spectrum
appeared as a broad signal at room temperature (25 1C). The
broad signal is a characteristic feature of self-assembled mole-
cules. Fig. 8 shows that the aromatic protons underwent shar-
pening with the increase in temperature. At the gel melting point
at 75 1C, the broad signals disappeared, and relatively sharp
peaks with multiplicity appeared. At this stage, the disassembly
of the peptide molecules occurred via disruption of p–p stacking
interactions, and the hydrogel turned into a solution. The
temperature dependent NMR study revealed that the p–p stack-
ing interactions play a vital role in the self-assembly process of
peptide bolaamphiphile 1 to form a hydrogel.73
2.7. X-ray diffraction study
In order to obtain advanced structural information, the dried
hydrogel of compound 1 was characterized by wide angle X-ray
scattering (WAXS). The scattering patterns show a series of
characteristic diffraction peaks (Fig. 9), from which we are able
to correlate the different features of the self-assembly process.
Fig. 7 (A) Decay traces of peptide bolaamphiphile 1 hydrogel (10 mmol Lꢀ1
)
at (ii) 15 1C, (iii) 40 1C and (iv) 80 1C temperature; (i) represents the IRF.
(B) Changes in the average lifetime of peptide bolaamphiphile hydrogel 1
with temperature.
Fig. 8 Temperature dependent 1H NMR spectra showing the p–p stacking
interactions during the self-assembly process.
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Fig. 11 Transmission electron microscopic image of peptide bolaamphiphile
hydrogel 1 showing that the peptide bolaamphiphile molecules self-assembled
into a nanofibrillar structure.
Fig. 9 Powder X-ray diffraction of solid powder of peptide bolaamphiphile
1 and its dried hydrogel.
microscopy (TEM). The TEM image shows that the peptide
bolaamphiphile molecules self-assembled into amyloid-like nano-
fibrillar networks which are responsible for formation of the
supramolecular hydrogel. The average diameter of the nanofibers
is 17 nm and each fiber is several micrometers in length (Fig. 11).
In the TEM image, there are some circular domains besides the
fibrillar structures. This indicates that the peptide bolaamphi-
philes self-assemble to form spherical nanostructures.
2.9. Characterisation of amyloid-like fibrils
Fig. 10 A model structure was created for peptide bolaamphiphile 1, which
is arranged in an anti-parallel b-sheet motif. Aromatic tryptophan moieties are
stacked by p-stacking interactions, and peptide chains are associated with
hydrogen bonding interactions, which results in the formation of nano-
structured fibrils.
Thioflavin T (ThT) is a positively charged dye, which is widely
used to monitor and detect the formation of amyloid-like
fibrils.79 ThT shows a very low fluorescence quantum yield in
low viscosity media like water because of its non-radiative bond
twisting process around the central C–C bond in the excited
state. Here, the binding of ThT (10 mM) with the peptide
bolaamphiphile 1 gel was monitored by means of spectral
changes in UV-Vis absorption and fluorescence spectroscopy.
The absorption spectrum of ThT in phosphate buffer shows a
peak at 412 nm. However, the peak shifted to 442 nm in the
presence of peptide bolaamphiphile hydrogel 1 with increase in
absorbance (Fig. 12A). The 30 nm red shift of the absorption
peak clearly suggests that ThT was strongly bound with the
b-sheet type structures resulting from peptide bolaamphiphile
1 in the gel state.80 Fig. 12B shows the fluorescence spectra of
ThT in the absence and presence of peptide bolaamphiphile
hydrogel 1. ThT shows very weak emission at 480 nm in phosphate
buffer on excitation at a wavelength of 412 nm. A significant
increase in fluorescence intensity has been observed for ThT in
peptide hydrogel 1 with a peak shift of 8 nm. We observed a
sixty-fold enhancement in fluorescence intensity for the hydro-
gel containing ThT compared to ThT alone in phosphate buffer.
Earlier, Banerjee et al. reported a similar kind of ThT binding
assay of an amyloid fibril forming peptide using absorption and
fluorescence spectroscopy.81 The significant enhancement in
the fluorescence intensity with 8 nm red shift strongly indicates
For the dried hydrogel of compound 1, the peak at 2y = 10.201
corresponds to a d-spacing of 8.67 Å, which is expected to be the
distance between two b sheets (inter-sheet distance). A char-
acteristic peak at 2y = 18.571 corresponding to a d-spacing of
4.77 Å, indicates the distance between two peptide molecules
within the b-sheet structure.74 Another two characteristic reflec-
tion peaks are observed at 3.86 Å (2y = 23.001) and 2.81 Å
(2y = 31.751). The 3.86 Å peak reveals the p–p stacking interactions
between two aromatic groups, and the peak at 2.81 Å suggests the
hydrogen bonding interactions between the CQO and N–H of the
peptide bolaamphiphiles.75
We propose a molecular model76 of the self-assembly mecha-
nism for the supramolecular hydrogel of peptide bolaamphiphile 1.
The model shows that the peptide bolaamphiphile molecules
self-assembled through hydrogen bonding interactions and p–p
stacking interactions between the tryptophan residues and form an
anti-parallel b-sheet structure (Fig. 10). These findings are well
correlated with the results of the FT-IR, fluorescence spectroscopy
and X-ray diffraction studies.
2.8. Transmission electron microscopy study
The nanostructural morphology77,78 of hydrogel 1 at the supra- the binding of ThT with the amyloid-like fibril forming82
molecular level was characterized using transmission electron peptide bolaamphiphile hydrogel 1. Nanofibrils resulting from
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been investigated. Hydrogen bonding interactions and p–p stack-
ing interactions are the driving force for the self-assembly process,
which results in the formation of a self-supporting hydrogel where
the peptide bolaamphiphile molecules adopt an anti-parallel
b-sheet structure. The self-assembly mechanism is influenced
by temperature and change in concentration, which was studied
by time dependent 1H NMR and time-resolved fluorescence
spectroscopy. Peptide bolaamphiphile hydrogel 1 turns to solution
with increase in temperature, which correlates with the results of
temperature dependent TCSPC studies. The average life time of
the peptide bolaamphiphiles decreases linearly with increase in
temperature. With increase in concentration, the peptide bola-
amphiphile molecules are organized in a more compact fashion
and form a b-sheet structure. The average life time of the peptide
bolaamphiphiles increases with increase in concentration up to a
certain limit, which was studied by concentration dependent
TCSPC measurement. The peptide bolaamphiphile molecules
self-assemble into a well-defined nanostructured morphology
at the supramolecular level upon sonication.
4. Experimental section
4.1. Synthesis of peptide bolaamphiphiles
Peptide bolaamphiphiles 1 and 2 were synthesized by conventional
solution phase methodology. The C-terminus of the amino acids
was protected as methyl ester. Couplings were mediated by dicyclo-
hexylcarbodiimide and 1-hydroxybenzotriazole (DCC-HOBt). The
final compounds were purified and fully characterized by FT-IR,
1H NMR, 13C NMR and mass spectral studies.
4.2. NMR spectroscopy
All NMR characterizations were carried out on a Bruker AV
400 MHz spectrometer at 300 K. Compound concentrations were
in the range 1–10 mmol in (CD3)2SO and CDCl3. The temperature
dependent NMR study was done in a phosphate buffer of D2O.
Fig. 12 (A) Binding of ThT into the amyloid fibrils of hydrogel 1 monitored by
spectral changes in absorption spectra (i) in phosphate buffer, and (ii) in peptide
bolaamphiphile 1 at 7 h. (B) Fluorescence spectra of ThT (i) in phosphate buffer,
(ii) in peptide bolaamphiphile 1 at 7 h, (iii) in peptide bolaamphiphile 1 at 1 day,
and (iv) in peptide bolaamphiphile 1 at 2 days. (C) Optical microscopic image of
peptide 1 fibrils stained with Congo red dye showing a green-gold birefrin-
gence, which is a characteristic feature of amyloid fibrils.
4.3. Mass spectrometry
Mass spectra were recorded on a Waters HPLCMS system
(Column Symmetry C18, 7 mm) by negative mode electrospray
ionisation and Bruker micrOTOF-Q II by positive mode electro-
spray ionisation.
the hydrogel of compound 1 were stained with a physiological
dye (Congo red) and viewed through a cross polarizer under a
polarized microscope (Fig. 12C). Fibrils of peptide hydrogel 1 bind
with Congo red and exhibit a characteristic green-gold birefringence
under the cross polarizer, which indicates amyloid-like behaviour.83
Thus, peptide 1 forms amyloid type fibrils in the hydrogel state, as is
evident from the typical green-gold birefringence after staining with
Congo red and the enhancement of fluorescence upon binding with
thioflavin T.
4.4. Polarimetry study
Specific rotations of the synthesized compounds were measured
on an Autopol V automatic polarimeter (Rudolph Research
Analytical). The cell (length = 100 mm, capacity = 2 mL) was
used for this study at 20 1C.
4.5. Rheology
Oscillating rheology was used to quantify the final mechanical
properties of the peptide bolaamphiphile hydrogels. In each
case, 2 mL of peptide bolaamphiphile hydrogel (10 mmol Lꢀ1
)
3. Conclusion
was prepared. The experiment was done on a Paar Physica
In this report, the self-assembly mechanism and photophysics Modular Compact Rheometer (MCR 301, Austria). A 25 mm cone
of amyloid-like fibril forming peptide bolaamphiphile 1 have plate with 11 angle configuration was used and the temperature
382 | New J. Chem., 2014, 38, 376--385
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was set to be constant at 25 1C. Storage (G0) and loss (G00) 4.11. Molecular modeling
moduli were measured at 0.1% strain with a true gap of
Models were constructed using the Hyper-Chem program. The
amber force field was used to minimize the energy of the
structures formed in this program.
0.05 mm. To determine the exact strain for the frequency sweep
experiment, the linear viscoelastic (LVE) regime was performed
at a constant frequency of 10 rad sꢀ1
.
4.12. Transmission electron microscopy
4.6. FT-IR spectroscopy
High resolution transmission electron microscopy images were
taken using a Philips electron microscope (CM 200), operated at
All reported FT-IR spectra were taken using a Bruker (Tensor 27)
FT-IR spectrophotometer. The solid-state measurements were
performed using the KBr pellet technique with a scan range
between 400 and 4000 cmꢀ1 over 64 scans at a resolution of
4 cmꢀ1 and an interval of 1 cmꢀ1. The gel sample was prepared
in D2O, placed between crystal Zn–Se windows and scanned
between 900 to 4000 cmꢀ1 over 64 scans at a resolution of
an accelerating voltage of 200 kV. The hydrogel (15 mmol Lꢀ1
)
was diluted to 0.75 mmol Lꢀ1 with double distilled water and
characterized using a transmission electron microscope. Dilute
solutions of the hydrogel were dried on carbon-coated copper
grids (300 mesh) by slow evaporation in air, and then allowed to
dry separately in a vacuum at room temperature.
4 cmꢀ1 and an interval of 1 cmꢀ1
.
4.13. Congo red binding study
4.7. UV-Vis spectroscopy
An alkaline saturated Congo red solution was prepared. The peptide
fibrils were stained with the alkaline Congo red solution (70%
methanol + 30% 0.08 M NaOH solution in double distilled
water) for 15 min, and then the excess stain (Congo red) was
removed by rinsing the stained fibrils with 70% methanol +
30% double distilled water solution several times. The stained
fibrils were dried at room temperature and then visualized at
100ꢁ magnification. The birefringence was observed between
crossed polarizers.
UV-Vis absorption spectra of the hydrogel were recorded using
a Varian Cary100 Bio UV-Vis spectrophotometer.
4.8. Fluorescence spectroscopy
Fluorescence and excitation spectra of the gels were recorded
on a Horiba Scientific Fluoromax-4 spectrophotometer with a
1 cm path length quartz cell at room temperature. The slit
width for the excitation and emission was set at 5 nm with a
1 nm data pitch. Excitations of samples were performed at
280 nm and 365 nm, and the data range was between 290 to 575
and 375 nm to 625 nm, respectively.
Acknowledgements
AKD is thankful to the DST (Grant no. SR/FT/CS-67/2010) and
CSIR (Grant no. 02(0056)/12/EMR-II), New Delhi, India for finan-
cial support. IM is indebted to CSIR, New Delhi, India for his
fellowship for this work. We thank the UGC-DAE Consortium for
Scientific Research, Indore for the use of powder XRD.
4.9. Time correlated single photon counting (TCSPC)
A 2 mL of gel sample was prepared in a quartz cuvette (1 cm ꢁ 1 cm)
and time correlated single photon counting (TCSPC) studies
were performed on a Horiba Yovin (model: Fluorocube-01-NL)
instrument. Samples were excited at 376 nm using a picosecond
diode laser (model: Pico Brite-375L). The signals were collected
at magic angle (54.701) polarization using a photomultiplier tube
(TBX-07C) as a detector, which had a dark count of less than
20 cps. The instrument response function (IRF, FWHM B 140 ps)
was recorded using a very dilute scattering solution. Data analysis
was performed using IBH DAS (version 6, HORIBA Scientific,
Edison, NJ) decay analysis software.
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