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Single amino acid based self-assembled structure†

Cite this: Soft Matter, 2013, 9, 10141
Shama Perween, Balasaheb Chandanshive, Hema Chandra Kotamarthi
and Deepa Khushalani*

Received 17th April 2013
Accepted 30th August 2013
DOI: 10.1039/c3sm51054a
www.rsc.org/softmatter
Single amino acids (phenylalanine, tyrosine and glycine) have been evaluated for fibrillar structure under
neutral, aqueous conditions using scanning electron microscopy, transmission electron microscopy, circular
dichroism, FTIR, and Congo red and thioflavin T histological dye assays. All these techniques prove that
aromatic amino acids, such as phenylalanine and tyrosine, do in fact form distinct fibrillar structures
albeit without any secondary structural characteristics such as an a-helix or a b-sheet. The nature of the
interactions between neighbouring amino acids in the fibrillar structures are purported to simply be
non-covalent p–p interactions.

Introduction desired. The brillar structure invariably is used as a structure-

Over the last two decades, literature citations have become
increasingly replete with ndings of new ways to design organic
structures with novel architectures and properties.1 Specically,
the self-assembly of peptides (with a variety of amino acid
sequences) and the applications of the ensuing structures in
various elds have been the focus of attention of many
researchers.2 One particular morphology that has gained a large
prominence in the literature is that of brils, specically b-
amyloid brils. In fact, b-amyloid forming peptides (that are
known to spontaneously form 1D structures) are one of the
main reasons that bres/brils/ribbons have garnered large
interest because they are known to play a pivotal role in a variety
of neurodegenerative disorders such as Alzheimer's disease and
phenylketonuria.3–6 These structures predominantly involve a
generic type of polypeptide conformation, termed a “cross-beta
structure”. This conformational state consists of intermolecular
b-sheets that transversely extend normal to the major axis of the
bril.
As a result, considerable work has been done on evaluating
the formation and morphology of self-assembled structures of
peptides, over a wide range spanning from polypeptides (that
may contain >10 residues) to the simplest in the family, the
dipeptides. In addition, from a materials chemistry point of
view, one of the main advantages of such spontaneous self-
assemblies arises from the fact that they have intriguing
hydrogelation properties that can be exploited in inorganic
materials synthesis where high aspect ratio structures are
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi
Bhabha Rd, Colaba, Mumbai, 400005, India. E-mail: Khushalani@tifr.res.in; Fax:
+91 22 22804610; Tel: +91 22 22782476
† Electronic supplementary information (ESI) available: SEM image for Phe at very
low concentration (<1 mm), TEM images for Phe, Tyr and Gly along with UV-Vis
data for Phe and Tyr are given. See DOI: 10.1039/c3sm51054a
directing template on which inorganic precursors are
condensed.7–11
In 2003, Reches and Gazit showed for the rst time that one
of the more simple self-assembling systems, a dipeptide
(phenylalanine–phenylalanine, FF), could form a bre-like
morphology.12 They showed tubular structures of the order of
several micrometers in length. These were prepared in mild,
aqueous conditions and were observed to possess a high aspect
ratio as well as high mechanical strength. These however were
not purported to form b-amyloid type structures. Reches and
Gazit also reported the self-assembling properties of FF resi-
dues with modied phenyl-side chains further conrming that
the homo-aromatic dipeptide moiety was indeed essential for
forming well-ordered 1D structures.13 Subsequently, many other
citations have appeared over the last decade detailing the
manner in which the FF dipeptide assembles and exploiting its
architecture for formation of hydrogels. These hydrogels are
purported to be useful in a wide variety of possible biomedical
applications including tissue engineering and controlled drug
release.14–17
An interesting report in 2010 by Ryan and co-workers
detailed using an even simpler biomolecule – namely a single
amino acid (phenylalanine, albeit FMOC protected with a per-
uorinated side chain, Fmoc-F5-Phe) – to form hollow tubular
structures.18 This Fmoc-F5-Phe displayed hydrogelation at
relatively low concentrations (<0.1 wt%) whereas they observed
that simple Fmoc-Phe (i.e. the un-peruorinated protected
amino acid) did not readily undergo self-assembly and hydro-
gelation. The difference observed was explained as being due to
perturbation of the side chain p–p interactions upon uorina-
tion. They concluded that hydrophobicity and sterics played
vital roles in aggregation.
Remarkably, in 2012 Adler-Abramovich and co-workers went
a step further and showed how even an unprotected single
amino acid (phenylalanine) could also in fact aggregate and

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form brillar structures.19 Their report detailed using a variety

of techniques including electron microscopy, diffraction and
thioavin T and Congo red assays to prove that phenylalanine
self-assembled into brils and moreover they opined that these
bres explicitly had amyloid-like morphology. As a consequence
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of this, in order to get further insight into the nature of inter-

actions between adjoining (unprotected and unfunctionalized)
phenylalanine molecules, presented here is a study of the self-
assembling properties of the single amino acid phenylalanine,
which forms bre-like structures in an aqueous medium at
neutral pH. Moreover, parameters such as concentration and
side chain functionality have been evaluated by extending the
study to two other amino acids, tyrosine and glycine.

Experimental section
Phenylalanine, tyrosine, and glycine were purchased from
Sigma and used without further purication. The purity of the
amino acids procured was 99.9%. Deionized water was used for
preparing all the solutions (Millipore). Scanning electron
microscopy (SEM) images were taken using a Zeiss Ultra FEG 55
microscope (the accelerating voltage ranged from 5 to 20 kV).
SEM samples were prepared on either silicon, glass, gold-coated
glass, mica or aluminium substrates by directly depositing 20
mL of the solution. Transmission electron microscopy (TEM)
images were captured on a ZEISS, LIBRA-120 machine operated
at 120 kV. Samples were dried on carbon-coated copper grids
(100 mesh). Circular dichroism (CD) spectra were recorded on a
JASCO J-810 spectropolarimeter. Fourier transform infrared
(FTIR) spectra of the samples were recorded on a JASCO FT/IR-
4100 spectrometer using a D2O and CaF2 liquid sample cell. For
the Congo red (CR) absorbance measurements, 4 mL of 1 mM
amino acid solution was incubated with 1 mL of 10 mM solution
of CR at RT. UV-Vis spectra of the resulting solutions were
recorded aer 30 min, 12 h and 24 h on a SPECORD 205 spec-
trophotometer. For thioavin T (ThT) photoluminescence (PL)
and confocal microscopy measurements, 1 mL of a 2 mM amino
acid solution was incubated with 1 mL of a 0.5 mM ThT solu-
tion. PL data were obtained aer 30 min, 12 h, and 24 h using a
Spex Fluorolog 1681 spectrometer and ThT was excited at 440
nm.
Results and discussion
Phenylalanine, tyrosine and glycine solutions were analyzed
within a concentration range of 1 mM to 100 mM (the upper
limit was set as some of the amino acids were not soluble at
concentrations higher than 100 mM). All the solutions were
prepared using only deionized water (pH ¼ 7) and no buffers
were employed. Fig. 1a displays a SEM image of the structures
formed in the 1 mM solution of phenylalanine. It can be seen
that they consist of long, bre-like aggregates. In the case of
Phe, it was found that bres could be observed only for 100 mM
and 1 mM. Lowering the concentration to 1 mM prevented the
self-assembling of any discernible structures (see Fig. S1, ESI†).
For the 1 mM concentration, the dimensions of the aggregates
were such that the width ranged from 300 nm to 800 nm and the
Fig. 1 SEM images of 1 mM solution of (a) L-Phe and (b) L-Tyr. Scale bar ¼ 2 mm.
length was on the order of several micrometers. These high
aspect ratio structures were observed even when the concen-
tration was increased up to 100 mM. However, above this
concentration, crystalline precipitates of phenylalanine were
observed. In addition, the SEM images were also reproduced
when the substrate was varied from silicon to glass, gold-coated
glass, aluminium or mica. This therefore directly suggested that
the bre-like structures that were forming were not due to
epitaxial interactions with the substrates and perhaps could
also exist as free standing bres in solution.
Fig. 1b displays an analogous SEM image for solutions
prepared with tyrosine. It can be observed that the product also
consists of straight bres (however much more so than with
phenylalanine), and the aggregation seems to provide well-
aligned structures with a at ribbon-like textural quality. For the
1 mM concentration, the dimensions of the aggregates were
such that the width ranged from 200 nm to 2 mm and the length
was on the order of several micrometers. Upon closer exami-
nation of the bres, Fig. S2 ESI,† it can be seen that for both Phe
and Tyr, the bres consist of a wide range of widths but are
homogeneously dense throughout their length. SEM and TEM
analysis was also performed on achiral glycine. No specic
morphology was observed at any concentration when observed
through either TEM or SEM analysis (see Fig. S3, ESI†).
Circular dichroism (CD) absorption is commonly used,
specically in the far UV-region (190–250 nm) to evaluate
secondary structure characteristics of proteins and amyloid
bres, and in the near UV-region (250–300 nm) to evaluate the

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tertiary structure of proteins.20 Fig. 2 displays the CD spectra of
phenylalanine, tyrosine and glycine. A visible absorption at 218
nm for phenylalanine and at 222 nm for tyrosine at 1 mM
concentration can be clearly observed, while glycine shows an
absence of any optically active signal. Normally for peptides, an
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absorption in the region of 190 to 260 nm is observed since the
peptide bond is a primary absorbing species.20 In this case,
despite the absence of any covalent amide linkages, the pure
amino acid solution appears to display secondary structure
characteristics that are manifested as absorption in the CD
measurement. Recently, Smith and co-workers reported a
similar absorption at 218 nm for Fmoc-FF and Fmoc-F and
suggested that this absorbance was consistent with a b-sheet
structure.21 However, reports in the literature detailed the origin
of this absorption band for Phe and attributed it to simple
transitions involving the carboxyl group (n–p*) and benzene
ring (p–p*), respectively.22–24 These transitions are also
observed for tyrosine albeit at a slightly different wavelength,
222 nm. For glycine, the n–p* transition occurs outside the far-
Fig. 2 Circular dichroism spectra of (a) Phe (b) Tyr and (c) Gly.
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UV region and so is not visible in this range. This assignment
therefore suggests that the far-UV absorption in a CD spectrum,
for at least single amino acids, is not always indicative of
secondary structure characteristics. Furthermore, in the near
UV region (250–300 nm) of Fig. 2, a weak signal is observed for
tyrosine (1 mM). This indicates an asymmetric environment
around the aromatic chromophore of tyrosine in the bre.
However, in the case of phenylalanine, there is no discernible CD
signal in the near UV region. In proteins, a CD signal in the
aromatic absorption region is typically interpreted as the presence
of an asymmetric environment around the aromatic residues
giving rise to a well-dened tertiary structure of the proteins.20
An additional, well-cited characterization tool for the
formation of a b-sheet structure is Fourier-transform infrared
spectroscopy. FTIR has been used to show that native b-sheet
proteins are associated with a very distinctive amide I band.
Amide I (measured in H2O) or amide I0 components (in D2O)
occur in the wavenumber range from 1600 cm1 to 1700 cm1
and are the most intense absorption bands. It is primarily
governed by the stretching vibrations of the C]O (70–85%) and
C–N groups (10–20%). The exact band position is determined by
the backbone conformation and the hydrogen bonding pattern
and hence it is sensitive to the secondary structural aspects and
dihedral torsional 4/f angles.25,26 To evaluate the nature of Phe,
Tyr and Gly structures in solution, transmission mode FTIR
spectra were recorded on samples prepared in D2O. The spectra
were obtained using a CaF2 liquid FTIR cell and recorded at
various concentrations. It was observed that there was an
absence of any discernable vibration mode in the region of 1600
to 1700 cm1 regardless of the type of amino acid used, its
concentration, or the temperature. This thereby gave a strong
indication that the b-sheet type of structure was absent in both
the Phe and Tyr bres. More importantly, this also provided
evidence that there was an absence of any covalent linkage
between neighbouring amino acids. Hence the self-assembly
process to yield bre like structures was based primarily on
weak interactions between the neighbouring amino acids.
Further analyses using Congo red (CR) dye absorption along
with thioavin T (ThT) photoluminescence measurements were
performed to get insight into the prevalent solution structure.
CR and ThT are both histological dyes that are regularly used as
indicators for the presence of amyloid structures. An initial
model of CR binding to brillar b-amyloids was developed by
Klunk and co-workers, whose preliminary experiments showed
that CR did not bind to a single b-amyloid peptide but actually
required the formation of a b-sheet containing bril.27,28
However, currently there is some debate as to the exact nature of
CR binding to amyloid bres (whether the alignment of CR is
perpendicular or parallel to the brillar direction), although it is
universally accepted that regardless of the manner of binding,
CR displays a red-shi in its absorption maximum, which in the
unbound form is normally observed at 497 nm. This shi has
been attributed to the expansion of p-conjugation in the phenyl
rings as they adopt a more planar conguration upon inter-
acting with a b-sheet structure.29 As for ThT binding, it has been
purported that the transition dipole of ThT lies parallel to the
long molecular axis of the brils. Therefore, ThT binds to
Soft Matter, 2013, 9, 10141–10145 | 10143

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amyloid brils such that their long axes are parallel and the
binding occurs in ‘channels’ that run along the length of the b-
sheet.30 Steric interactions between dye molecules and side
chains normally indicate that ThT uoresces more intensely
when bound to amyloid brils, although the exact nature of how
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specically it binds varies from bre to bre, and hence the
efficiency and the uorescence enhancement is qualitative
instead of being completely quantitative. Spectra obtained of
Phe and Tyr with CR and ThT are displayed in Fig. 3. Fig. 3a
displays the CR absorption spectra in the presence of the amino
acid solutions. A band with a maximum at 496 nm is observed
for the control CR solution. The position of this maximum and
its intensity are found to remain unchanged when CR is added
to the Phe, Tyr and Gly solutions. Moreover, the spectra remain
the same even aer ageing the solutions for up to 48 h or even if
the concentrations of the amino acid solutions are increased.
Fig. 3b displays the ThT photoluminescence spectra in the
presence of the amino acid solutions. In this case as well, an
emission maximum is seen at 485 nm and this also remains
unchanged (in intensity and position) upon varying the amino
acid solutions or their concentrations. These data therefore
directly correlate with the FTIR and CD studies given above and
specically suggest that the Phe and Tyr bres do not have
amyloid-type characteristics.
It should be noted that UV-Vis absorbance and PL emission
studies are quantitative methods that mirror what confocal
microscopy qualitatively provides. To give further credence to
the above data, additional confocal microscopy studies were
Fig. 3 (a) Absorbance spectra for solutions prepared with Congo red and Phe,
Tyr or Gly. (b) Emission spectra (excitation at 440 nm) for solutions prepared with
ThT dye and Phe, Tyr or Gly.
10144 | Soft Matter, 2013, 9, 10141–10145
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performed on ThT labeled glycine, phenylalanine and tyrosine
bres. Fig. 4 displays the confocal images for the three amino
acids. These studies were performed with due attention to the
ratio of ThT : amino acid such that all conditions were kept
identical to those used for obtaining the data already detailed
above. Excitation was performed at 390 nm. In Fig. 4a it can be
seen that under the focusing capability of a confocal micro-
scope, the solutions of Phe + ThT do not show any organized
structure. This aspect was repeatedly checked to ensure that the
solution phase was being imaged. Do note that at the resolving
power of an optical microscope, nanometer wide bres will not
be resolved. However, upon allowing some time to evolve, it was
observed that at the edges of the microscope slide, dehydration
of the solvent occurred, which led to formation of what appears
to be lamentous structures (Fig. 4b). Surprisingly, all three
amino acids show such morphology (including glycine). More-
over, even when pure ThT is imaged (i.e. no amino acid is
present in this solution), it also displays bre-like structures as
shown in Fig. 4e. If b-amyloid bres were present then ThT
would be able to intercalate within the layers and show not only
increased uorescence but also a shi in lmax. The confocal
Fig. 4 Confocal microscopy images of (a) ThT + 1 mM Phe solution, (b) fibres
observed at the edge of the microscope slide of ThT + Phe, (c) fibres observed at
the edge of the microscope slide of ThT + Tyr, (d) fibres observed at the edge of
the microscope slide of ThT + Gly and (e) fibres observed at the edge of the
microscope slide of pure ThT.
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images show that in fact ThT does not bind to any lamentous
structure. In fact it erroneously also gives an artifact – it
precipitates out at the edges of a microscope slide and does so
in bre-like structures.
Combining the information of all these spectroscopic tech-
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niques, it can be inferred that aggregation in Phe and Tyr can be
attributed to strong hydrogen bonds and electrostatic interac-
tions between the molecules. The stability of the bre form can
be accounted for by the obvious p–p interactions between the
phenyl residues of adjacent monomer molecules. Although
these interactions lead to 1D self-assembled structures, these
however do not have a b-sheet type of structure.
Conclusion
In summary, three amino acids (Phe, Tyr and Gly) have been
evaluated for their ability to form 1D brillar structures. It has
been observed that only the two amino acids with aromatic side
chains are capable of spontaneously forming self-assembled
structures in solution, for which a minimum concentration of 1
mM is needed. Moreover, these brillar structures do not exhibit
any secondary structure characteristics such as a b-sheet,
although they have relatively strong intermolecular non-cova-
lent p–p interactions.
Acknowledgements
We gratefully acknowledge Ms G. Ramasubraminiam with
assistance in the earlier stages of this project. In addition, we
thank the TIFR Cryo-TEM facility, Mr L Borde and Ms B Chalke's
assistance with EM measurements.
Notes and references
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