[System] polypeptide self-assembly literature information extraction

[User]
We explore the gel transition of the aromatic dipeptide derivative molecule
fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF). The addition of water to a
solution of Fmoc-FF in dimethyl sulfoxide (DMSO) results in increased
attractions leading to self-assembly of Fmoc-FF molecules into a space-filling
fibrous network. We provide evidence that gel formation is associated with a
first order phase transition resulting in nucleation and growth of strongly
anisotropic crystals with high aspect ratios. The strength of attraction between
Fmoc-FF molecules as a function of water concentration is estimated from
long-time self-diffusion measurements using (1)H NMR diffusion-ordered
spectroscopy (DOSY). The resulting phase behavior follows that observed for a
wide range of other crystallizing nanoparticles and small molecules - a result
consistent with the short-range nature of the intermolecular attractions.
Furthermore, we use NMR to measure the rate of increase in the fraction of bound
Fmoc-FF molecules after water is suddenly mixed into the system. We observe a
lag time in the formation of the new phase indicative of the existence of a free
energy barrier to the formation of a crystal nucleus of critical size. The
application of classical nucleation theory for a cylindrical nucleus indicates
that one-dimensional crystal growth is driven by an imbalance of the surface
energies of the ends and sides of the fiber.

We explore the gel transition of the aromatic dipeptide derivative molecule fluorenylmethoxycarbonyl-
diphenylalanine (Fmoc-FF). The addition of water to a solution of Fmoc-FF in dimethyl sulfoxide (DMSO)
results in increased attractions leading to self-assembly of Fmoc-FF molecules into a space-filling
fibrous network. We provide evidence that gel formation is associated with a first order phase transition
of attraction between Fmoc-FF molecules as a function of water concentration is estimated from long-time
self-diﬀusion measurements using 1H NMR diﬀusion-ordered spectroscopy (DOSY). The resulting phase
behavior follows that observed for a wide range of other crystallizing nanoparticles and small molecules – a
measure the rate of increase in the fraction of bound Fmoc-FF molecules after water is suddenly mixed into
the system. We observe a lag time in the formation of the new phase indicative of the existence of a free
Molecular gels are formed from the self-assembly of molecules
experiencing hydrogen bonding and p–p stacking interactions
into anisotropic structures, typically fibers of high aspect ratios,
which produce space-spanning networks that resist shear,
resulting in a bulk material with properties characteristic
of soft solids.1,2 A specific class of molecular gels that has
gained considerable attention over the past decade are self-
assembling systems based on short peptide derivatives, which
form ordered fibrous structures, usually based on p–p stacking
of b-sheets.3 The most widely studied such molecule is the dipeptide
fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF).4–12 Addition
of water to low-concentration solutions of this molecule in
dimethyl sulfoxide (DMSO) results in the formation of mechanically
and thermally reversible gels with long relaxation times and
substantial elastic moduli.9–12 Due to ease of chemical modifi-
cations, Fmoc-FF and similar gelators provide opportunities to
design materials with unique structural, optical, mechanical and
biological functionalities.3 The gelation mechanism in these types
of systems remains poorly understood, limiting the general design
of new molecules and the engineering of new products based on
these molecular gels for applications in tissue engineering, drug
delivery, nanofabrication, biosensing, etc.13–16
Here we provide evidence that, when exposed to water,
Fmoc-FF molecules in DMSO undergo a first order phase
transition to form crystalline fibers of high aspect ratios. This
crystalline morphology arises from anisotropic molecular inter-
actions that give rise to substantial diﬀerences in surface
tension at the ends and sides of the resulting crystals. Due to
limitations of rates of mixing, upon initial addition of water,
very high strengths of attraction are achieved, resulting in the
formation of a metastable phase that is subsequently displaced
by fiber formation.17 This initial phase is amorphous, and the
ensuing formation of an ordered phase indicates that the
crystalline phase has the lower free energy. Our results suggest
that the soft solid mechanical response of the gels arises from
the low number density of intermolecular (physical) bonds, and
the ease of repairing structures damaged by mechanical disruption
through rapid dissolution and recrystallization processes is
enabled by high surface areas of the crystals.
To build the case that Fmoc-FF molecules undergo a first order
phase transition in forming fibers, we first seek to move from a
description of gel line denoted by concentrations of water and
Fmoc-FF at the gel point (Fig. 1) to a phase diagram where water
concentration is converted to an eﬀective strength of intermolecular
interaction and a phase diagram where transitions can be denoted
Gelation in colloidal and protein solutions has been associated
with rapid quenches into the spinodal of metastable liquid–liquid
phase boundary that exists for particles experiencing short-range
attractions. The increased mechanical strength of the gels is
associated with low particle diffusivity in the dense, amorphous
state resulting in aggregation of clusters into space-spanning
structures. Aging and ultimate crystallization of these gels is
then expected (and observed) as the system slowly relaxes into
the lowest free energy minimum of a crystal in equilibrium with
a dilute molecular solution. The key features we draw from this
experimental and theoretical work are:
(1) When molecules experience short-range attractions, as
the strength of attraction is increased or the temperature is
lowered, molecules will spontaneously phase separate into
crystalline and dilute amorphous phases.
(2) At the same average strength of attraction, broad classes
of molecules have the same solubility and a coexistence region
exists that follows a lever arm rule relating the average concentration
of the solute to the volume of the two equilibrium phases.
We explore the application of these ideas to the Fmoc-FF/
DMSO/H2O system. As the water concentration is decreased,
higher Fmoc-FF concentrations are required to see a gel (Fig. 1).
Furthermore, at fixed Fmoc-FF volume fraction, increasing the
amount of water in the system results in increased elasticity.
These observations clearly indicate that water is responsible for
increased attractions between the Fmoc-FF molecules and that
there is a change of state of the Fmoc-FF molecules over a
narrow change in water concentration. Previously, we demonstrated
that the Fmoc-FF gel is reversible with respect to yielding upon
intense mechanical agitation and melting with increases in
temperature.17,32 The fibers are observed to form and fluctuate
in local concentration over relatively short periods of time
(minutes to hours).33
Fmoc-FF is hydrophobic and insoluble in water, but dissolves
to very high concentrations in DMSO (volume fractions
approaching 0.5), resulting in stable solutions. In the presence
of water, the peptide backbones form hydrogen bonds necessary
to create beta sheets, while the aromatic nature of fluorenyl
group and the amino acid side groups allow for p–p stacking
interactions,5 which are highly anisotropic and short-range
relative to the size of the Fmoc-FF molecule. Compact or chunky
crystals are not formed when water is added to an Fmoc-FF/
DMSO solution. Nevertheless, the produced fibers are ordered,5
suggesting that they can be treated as an equilibrium crystalline
state formed from anisotropic particles experiencing short-range
in terms of the strength of attraction between Fmoc-FF molecules
and the Fmoc-FF concentration. With this information in hand, we
demonstrate that the phase boundary of this molecule can be
mapped onto generalized phase behavior seen for a variety of
molecules and colloidal particles that experience isotropic and
weakly anisotropic attractions. We further demonstrate that at
constant strength of attraction, two phases are in equilibrium as
the average concentration of the Fmoc-FF molecule is increased
above the phase transition concentration. Finally, we show that
there is a weak barrier to crystal nucleation and discuss this barrier
in terms of the quasi-one-dimensional crystal nucleation that takes
place in the studied system.
Below, we first describe theoretical and experimental studies of
the phase behavior of small molecules and nanoparticles and how
diﬀusivities can be used to characterize strength of interparticle
attractions. We then proceed to describe our experimental
methods and discuss our results where we link diﬀusion to
strength of attraction and strength of NMR signal to freely
diﬀusing molecule concentration. Finally, we draw conclusions
about the phase behavior of the gelator in the Fmoc-FF/DMSO/
H2O system and propose the appropriate phase diagram.
Detailed theoretical, simulation and experimental studies suggest
that short-range isotropic attractions result in a phase diagram
where liquid–liquid phase separation is always metastable
relative to a fluid-crystal phase boundary.18–20 The spinodal
defining a dilute-concentrated or liquid–liquid phase separation
(analogous to a gas–liquid phase transition for single component
systems) is metastable and occurs at concentrations larger
(temperatures lower) than the crystallization boundary. Studied
extensively for colloidal particles and proteins (diameters
greater than 50 nm), the generalized nature of these phase
transition boundaries has been extended to small molecules
and proteins experiencing anisotropic interactions.21–27 The
presence of the metastable liquid/liquid phase separation has
been seen in proteins and small molecule systems. These studies
suggest that at the same average strength of attraction, broad
classes of Brownian particles display very similar solubilities.27–30
interactions. We explore this hypothesis by comparing the phase
behavior of the Fmoc-FF/DMSO/H2O system to other systems
experiencing generalized phase behavior.
can be used to measure diﬀusion of small molecules, as well as its
ability to provide signals of diﬀerent molecules in the solution
even at low concentrations.
This is done in several ways. First, we make a connection to
previous studies showing that a wide variety of organic and
inorganic molecules show very similar phase behavior when
their solubilities are compared at the same average strength of
attraction. Second, we show that the lever arm rule is operational
in the gelled state. Finally, we measure the induction time for the
formation of the fiber phase and again show consistency with
classical nucleation theories used to describe phase transitions of
small molecules and colloids. Each of these studies provides
evidence that the Fmoc-FF/DMSO/H2O system undergoes a first
order phase transition to form a highly anisotropic crystalline
phase and that the large clusters observed in solution on rapid
mixing with water are indicative of a quench into the spinodal of
a liquid–liquid phase separation that relaxes with time towards
an ultimate equilibrium state.
The volume fraction dependence of the long-time self-diﬀusivity
of Fmoc-FF carries information about molecular interactions.
The molecules diﬀuse more slowly if they spend time in close
proximity where they are more strongly hydrodynamically
coupled. Intermolecular attractions increase the time molecules
spend at small separations, thus further slowing the rate of
diﬀusion. In the dilute limit, assuming the molecules are
spheres, the long-time self-diﬀusion coeﬃcient, DS, is a linear
function of molecule volume fraction:34–36
where D0 = kT/6pma is the Stokes–Einstein diffusivity assuming
no-slip solid/fluid boundary conditions, f is the molecule
volume fraction, while D2 reflects hydrodynamic interactions
between pairs of particles. If the particles experience attractions,
the pair distribution will be altered such that pairs spend more
time at smaller separations. Stronger hydrodynamic coupling at
small separations results in smaller values of D2 (i.e. D2 becomes
more negative). As discussed later on, we assume that under the
dilute conditions, the particle pair distribution function is well
approximated as g(r) = exp[(cid:2)u(r)/kT] for r 4 s, where u(r) is the
pair interaction energy for particles of diameter s with center-to-
center separation r. For u(r) we use a square well potential and
assume the particles are spheres and interact with hydrodynamic
mobility functions Aij(r) and Bij(r) for particles experiencing no-slip
boundary conditions34,35 (see ESI†). With these approximations we
are able to use measurement of volume fraction dependent
diffusivities to extract an estimate of interparticle attraction.
As the particles experience anisotropic interactions, but are
Brownian, we expect the average strength of attraction extracted
from our approximations to be weighted towards the largest
strength of attraction.
While we determine D2 in the dilute limit where DS is a linear
function of f, we investigate translational diﬀusion in Fmoc-FF
solutions at conditions approaching and above the gelation
boundary and measure the gelation kinetics of slowly gelling
samples using 1H NMR spectroscopy. We use the changes in static
1H NMR signal intensity over time as a measure of the conversion
of Fmoc-FF molecules into solid structures in the gel. Using the
obtained data, we explore the hypothesis of Fmoc-FF fibers being
an equilibrium crystalline state and develop a phase diagram for
the Fmoc-FF/DMSO/H2O system using simple fluid diﬀusion
models and crystallization theories.
Solid Fmoc-FF powder was purchased from Bachem (Bubendorf,
Switzerland) and used without further purification. Dimethyl
sulfoxide (99.5%, Sigma-Aldrich) and Milli-Q water were used
in sample preparation for rheology measurements. For NMR
measurements, deuterated solvents were used (deuterium
oxide, D2O, and deuterated DMSO, (CD3)2SQO), obtained from
Cambridge Isotope Laboratories Inc. For clarity and consistency
purposes, the deuterated solvents will be referred to simply as
DMSO and H2O throughout the text. Fmoc-FF was dissolved in
DMSO at the desired volume fractions, calculated by:
All NMR measurements were performed on a 750 MHz Agilent
VNMRS NMR spectrometer, equipped with a 5 mm Varian
1H{13C/15N} PFG XYZ probe. The instrument was operated
in liquid-state mode and controlled using VNMRJ software
v3.2A. Compositions near the gel point were chosen at water
concentrations xH2O = 0.25–0.40 (Fig. 1), ensuring a sufficiently
long gelation time to allow mixing and transfer to the NMR
tubes (Z0.5 hours).
The solution was mixed with deuterium oxide and 700 mL
was transferred into a 5 mm thin wall precision NMR sample
tube (Wilmad-LabGlass). The sample tube was immediately
placed into the NMR instrument and the collection of 1H
spectra at selected time intervals was commenced. The first
1H spectrum was taken as the reference of the fully liquid state
at time t = 0 for each sample. The final conversion to solid of
each sample was determined from the 1H spectrum taken after
Fig. 2 1H DOSY spectra of Fmoc-FF in pure DMSO (fFmoc-FF = 0.027). The peaks shown correspond to the aromatic protons of the molecule (see Fig. S1,
ESI† for full spectrum).
The self-diﬀusion coeﬃcients were measured using the DOSY
capabilities of the 750 MHz Agilent VNMRS instrument. The
experiments were performed with liquid solutions at various
Fmoc-FF volume fractions in both pure deuterated DMSO and
solutions with deuterated water concentrations xH2O r 0.40.
PFGSE NMR utilizes the attenuation of the echo signal from a
spin-echo pulse sequence containing a magnetic field gradient
pulse at each period to determine the displacement of the
observed spins on timescales of milliseconds to seconds.38,39
The self-diﬀusivity of the spins can then be correlated to the
attenuation of the spin echo signal (see ESI† for technique details).
Rheology experiments were performed on a TA Instruments
DHR-3 rheometer with a Peltier plate and solvent trap, using a
41/40 mm cone geometry. Fmoc-FF/DMSO solutions were prepared
as previously described. As soon as water was added to the
solution in a vial, the sample was mixed lightly and 1.2 mL of
the mixture was quickly transferred onto the rheometer. The
rotating element was lowered into the measurement position
and the experiment was started immediately. The evolution of
the elastic modulus with time was measured at 25 1C, at an
oscillating strain of 0.005 and frequency 1 Hz.

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[System] polypeptide self-assembly literature information extraction

[User]
Peptide co-assembly is of interest for the development of functional
supramolecular biomaterials. Herein, computational simulations were combined
with experimental validation to aid the design and understanding of cooperative
co-assembly of a structure-forming tripeptide (FFD) and a functional
copper-binding tripeptide (GHK) leading to hydrogel formation in response to
complexation with copper ions.

Supramolecular self-assembly1,2 provides simple functional
mimics of biological materials.3–5 Co-assembly of two or more
components provides options for control of morphological,
physical and mechanical properties where the co-assembled
material may have properties that are not found in the individual
components. This cooperativity may be achieved by combining
low molecular weight gelators with polymers6 or surfactants;7
or (aromatic) peptide amphiphiles with surfactants,8–10 poly-
saccharides,11,12 proteins13,14 or light responsive gelators.15
Co-assembly in these systems is typically governed by non-
covalent interactions, in particular electrostatics.
While most existing supramolecular peptide biomaterials
are based on long peptides,16 typically containing eight or more
amino acids, it is increasingly recognized that di-17,18 or tripeptides
contain suﬃcient molecular information to enable formation
of self-assembling materials, including hydrogels,19–22 tunable
emulsions,23 stable surface coatings24 and most recently, supra-
molecular substrates for formation of polymeric pigments with
tunable properties.25 Thus, supramolecular structures based on
short peptides hold promise as functional biomaterials.26–29
a WestCHEM/Department of Pure and Applied Chemistry, University of Strathclyde,
295 Cathedral Street, Glasgow G1 1XL, UK.
E-mail: Yousef.abul-haija@glasgow.ac.uk
b WestCHEM/School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK
c Advanced Science Research Center (ASRC) at the Graduate Center of the City
University of New York (CUNY), New York, NY 10031, USA.
E-mail: rein.ulijn@asrc.cuny.edu
d Department of Chemistry and Biochemistry, Hunter College of CUNY,
695 Park Ave., New York, NY 10065, USA
e PhD programs in Biochemistry and Chemistry, The Graduate Center of the City
University of New York, NY 10016, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc04796g
A number of short peptides are known to possess specific
biological functions. These short peptides have been eﬀectively
introduced into supramolecular biomaterials through covalent
linking to the structure-forming component of the system.
Examples include materials bearing the fibronectin-derived
RGDS,30,31 or RGD32,33 the laminin-derived IKVAV34 the tyrosine
kinase nerve growth factor receptor-derived KFG.27 and the
catalytically relevant35 histidine containing tripeptides (such as
HFF).36 Another example is GHK, which is a copper-binding
extracellular matrix derived peptide sequence that plays a role in
wound healing.37,38
In this communication, we demonstrate how non-covalent
incorporation of short functional peptides (exemplified by glycine–
histidine–lysine, GHK) into supramolecular structures can be used to
functionalise short peptide hydrogels. In particular, this work
focuses on the design and co-assembly of a structure-forming
tripeptide (diphenylalanine-aspartic acid, FFD)21,23 and GHK leading
to hydrogel formation in response to complexation with copper ions.
GHK is itself not a gelator, and FFD and DFF were selected
as candidate structural tripeptides due to their previously
described self-assembly and charges that complement that of
GHK. FFD and DFF were previously shown to form bilayer-like
aggregates, but do not form gels in isolation at neutral pH (FFD
has been shown to form a hydrogel at pH 5).23
30 mM of GHK and 30 mM of FFD were dissolved separately
in PBS buﬀer (pH 7.4) at room temperature. Samples were
sonicated and vortexed in order to improve solubility and
homogeneity. Both samples formed clear solutions. To investi-
gate whether electrostatic interactions of the positively charged
lysine (K) and the negatively charged aspartic acid (D) could
induce co-assembly, a mixture of GHK/FFD was prepared at the
same conditions (i.e. 30 mM of each, 60 mM total) which
formed a clear solution (no hydrogel was formed). Interestingly,
spontaneous hydrogel formation was observed in the presence
of 30 mM CuCl2, suggesting that GHK complexation with
copper ions leads to the co-assembly and gelation observed.
Fig. 1 TEM images of all tripeptides showing random aggregates of GHK
(A), self-assembly of FFD nanofibers (B), nanotape formation due to the
co-assembly of FFD/GHK (C) and nanofiber formation due to the co-assembly
of FFD/GHK after complexation with copper ions (D). Insets show the optical
images of the corresponding peptides in inverted vials.
formed random aggregates, Fig. 1A. FFD (which was previously
observed to form bi-layered nanotubes in acidic conditions)23
formed nanofibers at pH 7.4, Fig. 1B. Upon mixing of both FFD
and GHK, the peptides co-assemble to form tape-like structures,
Fig. 1C. However, this co-assembly did not lead to gelation as is
typically observed for 2D self-assembled structures, such as
tapes and sheets39 that are usually not able to entrap the solvent
(in this case water) within their networks. When CuCl2 was
mixed with the FFD/GHK mixture, networks of nanofibers were
formed leading to hydrogel formation, Fig. 1D. The TEM images
suggest a rapid nucleation and growth mechanism giving rise to
spherulite-like structures.40 Additional TEM images with diﬀerent
magnifications are presented in Fig. S1 (ESI†).
Having established the co-assembly conditions for the
successful formation of hydrogels, we used computational
simulations to provide further understanding of the molecular
results were obtained using
co-assembly. Computational
GROMACS 4.5.3 utilizing the MARTINI coarse grain (CG) force
field. In each system, 300 peptide molecules are added (150 of
each for mixed systems) to a 12.5 nm3 box solvated with MARTINI
coarse grained water. This gives an approximate peptide concen-
tration of 200 mM, which is a factor of 10 greater than normal
experimental conditions, consistent with previous work in this
field where the increased concentration is required to accelerate
the simulated aggregation process.21,41,42 Each simulation is
equilibrated for a total of 9.6 ms, with snapshots taken at initial,
mid-point and final stages, Fig. 2. As can be seen, for GHK
(Fig. 2A) the peptides show some clustering but overall maintain
a random arrangement. For FFD, (Fig. 2B), the peptides arrange
themselves with the phenylalanine rings interacting with aromatic
side chains of other peptides. This induces the formation of a
bilayer structure, with a hydrophobic core (FF) and negatively
charged hydrophilic (D) surface. This formation is already
observed at the start of the simulation, indicating a rapid
hydrophobic collapse. When introducing GHK into a FFD system
(Fig. 2C) a co-assembly is observed where GHK peptides are
organized on the surface of the FFD structure. The computational
results suggest that this occurs due to the electrostatic interaction
between the lysine and the anionic surface of the FFD.
To assess the eﬀect of hydrogen bonding interactions
experimentally, and to assess the formation of salt bridges
between D and K residues, FTIR measurements were performed
in D2O for individual components (GHK and FFD) and for
mixtures of both before and after complexation with copper
ions, Fig. 3A. For both GHK and FFD broad FTIR peaks at
around 1560 cm(cid:2)1 is assigned for the deprotonated COO(cid:2). The
shift of this peak towards a lower wavenumber for FFD/GHK is
indicative of the formation of more ordered structures with
possible salt-bridge formation in co-assembled mixtures.21,23 It
is worth noting that, although complexation with Cu2+ leads to
fiber/hydrogel formation, the FTIR spectrum does not show a
clear shift of peak at 1560 cm(cid:2)1. This observation might be
explained as that salt-bridge formation becomes less significant
after complexation with copper ions, thus reducing the eﬀective
salt-bridge. The appearance of the peaks in the amide I region
indicated the presence of hydrogen bonding between the amide
Fig. 3 (A) FTIR spectra of tripeptides in isolation and after co-assembly,
samples (30 mM of each component) were prepared in deuterated
phosphate buﬀer pH 7.4 at room temperature. (B) Rheology data for the
FFD/GHK–Cu hydrogel with associated storage and loss moduli.
carbonyls. However, the lower intensity of these peaks suggest
that these are weak hydrogen bonds. As these peaks are present
in only samples containing the copper, we can conclude that
the presence of the copper helps the assembling propensity of
the tripeptides resulting in hydrogelation, whilst
highly ordered nanostructures. There is also a shift in the
signal from FFD in the presence of both GHK and copper
which indicates the formation of more stable structures.
In order to assess the role of copper ions in specific binding
and hydrogelation, we studied the gelation ability of GHK/FFD at
diﬀerent conditions. For instance, Cu2+ concentrations were varied
(i.e. 20, 30, 40, 50 mM) and diﬀerent metal ions (Zn2+, Co2+) were
used as summarized in Table S1 (ESI†) (all added as chloride salts).
We observed that gelation requires stoichiometric Cu2+ ions concen-
tration (30 mM) and the gel was not formed when a lower Cu2+ ion
concentration (20 mM) or much higher concentration (50 mM),
most probably due to super saturation of the peptide-copper com-
plexes in solution, thus preventing assembly. The lack of gelation for
Zn2+ and Co2+ demonstrates that GHK binds specifically to Cu2+ 43,44
and this is required to enhance the peptide co-assembly leading to
hydrogel formation. As previously reported, single crystal X-ray
diﬀraction studies revealed that copper ions binding to GHK involves
the N-terminal group of glycine, the nitrogen atom of the first amide
bond, the unprotonated imidazole nitrogen from histidine, and two
oxygen atoms in water (see also Scheme S1, ESI†).45
Having established the co-assembly conditions and ion-
selectivity of gelation, we studied the mechanical properties
of the formed hydrogels by rheology as shown in Fig. 3B. The
rheology data shows that the hydrogel formed at 30 mM Cu2+
has an average loss modulus value of 1.5 kPa, while the
hydrogel formed at 40 mM Cu2+ has a weaker gel with a storage
modulus of about 0.7 kPa. The slope of the modulus might
indicate thickening instability of the sample when 30 mM of
CuCl2 was used as previously reported for similar systems.46
To assess the importance of peptide sequence, we also
studied the co-assembly process in the presence of DFF instead
of FFD, Fig. 4. Consistent with previously reported structure
forming tripeptides (i.e., anionic amino acids are preferred at the
C-terminus),21 no gel formation was observed for individual
components as well as for mixtures before and after the addition
of copper ions, Fig. 4A–C. TEM images (Fig. 4D–F and Fig. S2,
ESI†) also suggest that both individual peptide solutions and
mixtures are unable to self-assemble to form highly organized
structures both in the presence and absence of copper ions. These
results are in good agreement with our previous21 and current
computational predictions (Fig. S3, ESI†).
Molecular dynamics simulations of DFF and GHK mixtures
show clear diﬀerences when compared with FFD/GHK, Fig. S3
(ESI†). The DFF structure compared with the FFD shows similar
bilayer formation. The introduction of GHK appears to cause
the collapse of the DFF structure resulting in random aggregates
of DFF/GHK, as a result, no hydrogelation is observed in the
presence of Cu2+. Similarly, MD simulations of FDF self-assembly
have resulted in a structure formation similar to that of DFF and
FFD (Fig. S4, ESI†). However, the presence of aspartic acid in the
middle position in the co-assembled system disrupts the packing
Fig. 4 Optical images (upper panel) and TEM images (lower panel) of the
control (DFF) aggregation (A and D), mixed with GHK (B and E) and
co-assembled sample after complexation with copper ions (C and F).
of the FF dyad within the tripeptide. The loss of the intra-
molecular p-interactions between the phenyl groups indicates
that the FDF structure is not stable enough to form an elongated
fibrillar structure when GHK is present.
It is worth noting that GHK has generated significant inter-
est from the cosmetics industry, in particular as a complex with
Cu2+ which facilitates copper intake by cells to enhance enzymatic
processes, wound healing and promotes angiogenesis.47,48
It also improves skin firmness and texture, fine lines and
hyperpigmentation.37,38 Thus, there is clear evidence of the
biological activity of GHK and GHK–Cu complex and it is
therefore considered as an additive for cosmetics and skin
treatments. Formulating GHK into a biocompatible tripeptide
hydrogel may be of direct interest for cosmetics industry.
In summary, the cooperative co-assembly of functional and
structural tripeptides has been studied by combining both
experimental validation and computational simulations. We have
found that the co-assembled peptides can selectively complex with
copper ions, leading to structural reconfiguration from clear solution
of nanotapes to hydrogel formation of nanofibers. GHK is a known
bioactive peptide and the hydrogels developed here may have
applications in wound care and cell culture, which will require
further investigation. The combined computation/experimental
approach followed here provides a generally useful approach to
identification of co-assembled structures of structural and functional
components based on short peptides.

[Assistant] [{"peptide_sequence": "FFD", "self-assembly_phase": "fiber", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "12.82", "PH": "5.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FFD", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "12.82", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "DFF", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 PBS", "peptide_concentration(mg/ml)": "12.82", "PH": "7.4", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
We have investigated the self-assembly of fluorenylmethoxycarbonyl-conjugated
dialanine (Fmoc-AA) molecules using combined computational and experimental
approaches. Fmoc-AA gels were characterized using transmission electron
microscopy (TEM), circular dichroism (CD), Fourier transform infrared (FTIR),
and wide-angle X-ray scattering (WAXS). Computationally, we simulated the
assembly of Fmoc-AA using molecular dynamics techniques. All simulations
converged to a condensed fibril structure in which the Fmoc groups stack mostly
within in the center of the fibril. However, the Fmoc groups are partially
exposed to water, creating an amphiphilic surface, which may be responsible for
the aggregation of fibrils into nanoscale fibers observed in TEM. From the
fibril models, radial distribution calculations agree with d-spacings observed
in WAXS for the fibril diameter and π-stacking interactions. Our analyses show
that dialanine, despite its short length, adopts a mainly extended polyproline
II conformation. In contrast to previous hypotheses, these results indicate that
β-sheet-like hydrogen bonding is not prevalent. Rather, stacking of Fmoc groups,
inter-residue hydrogen bonding, and hydrogen bonding with water play the
important roles in stabilizing the fibril structure of supramolecular assemblies
of short conjugated peptides.

Preparation of Fmoc-AA gels—Fmoc-Ala-Ala-OH was purchased from Bachem, Inc.
(Torrance, CA) and used without further purification. To form gels via a pH trigger, Fmoc-
AA was added to deionized water, vortexed, and sonicated for several minutes. 1 eq 0.5 M
NaOH was added and mixture was vortexed until completely clear. The mixture was then
added quickly to a vial containing 1 eq 0.1 M HCl, to trigger gelation.
Wide angle X-ray scattering—Fmoc-AA gel samples were prepared at 10 mg/ml (final
pH = 2.6–2.8) by a pH trigger method (1 eq 0.5 M NaOH added to dissolve Fmoc-AA, then
1 eq 0.1 M HCl added to induce gelation, as described above) and were spread out onto a
quartz slide and allowed to dry for two days. Samples were run on a Scintag X1 theta-theta
powder diffractometer with a Cu x-ray source. Peak analysis was performed using Jade
v9.1.1 (Materials Data Inc.) software, and d-spacings were calculated assuming an x-ray
wavelength of 1.54059Å (Cu K-α1).
Transmission Electron Microscopy—Gels for TEM analysis were prepared at a final
concentration of 5 mg/ml (final pH = 3.3–3.4). Approximately 10 μl of gel was placed on
the shiny side of Formvar/Carbon coated 300 mesh nickel grids (Electron Microcopy
Sciences, Hatfield, PA) and was allowed to adsorb for 2 minutes. Excess solvent was
removed using a Kimwipe, and the grid was further allowed to air dry for 1 minute, after
which the grid was washed with 10 μl deionized water for 1 minute and dried with a
Fourier Transform Infrared spectroscopy—Gels were prepared at 5 mg/ml (final pH
= 3.3–3.4) as described above, except D2O was used in place of deionized water in all
solutions. For pH triggered gels, NaOH and HCl solutions were diluted to the concentrations
described above using D2O. Gel samples were sandwiched tightly between two calcium
fluoride (CaF2) windows (25 mm wide by 2 mm thick) and placed in line with the beam in a
transmission configuration within the instrument. A Thermo-Mattson Infinity Gold FTIR
instrument was used to collect spectra, which were averaged over 200 scans.
Circular Dichroism spectroscopy—Fmoc-AA gels for circular dichroism were
prepared at a concentration of 5 mg/ml. ~2.5 eq of Glucono-delta-lactone (GdL), a slowly-
hydrolyzing lactone that ring-opens to form gluconic acid, was used to acidify and hence gel
a solution of Fmoc-AA and 1 eq 0.5 M NaOH in deionized water, as described previously.27
Prior to GdL addition, a background spectrum was taken using deionized water with 1 eq 0.5
M NaOH added. After GdL addition and dissolution the Fmoc-AA solution was pipetted
into a cylindrical demountable quartz cuvette with a 0.1 mm pathlength. Spectra were
measured using a Jasco J-810 CD Spectrometer every 10 minutes after GdL addition. The
instrument was operated with a data pitch of 0.5 nm, scan speed of 200 nm/min, and
response time of 1 second. Three spectra were taken for each time point then averaged. After
the final time point we checked for linear dichroism (LD) artifacts by performing the scan
again with the cuvette rotated 90, 180, and 270 degrees from its starting position, but we
observed no significant change in the CD signal and thus concluded that LD effects were
minimal. The final pH of GdL-formed gels after 24 hours was in all cases between 3.5 and
Initial structures preparations—Fmoc-AA molecules were built in TINKER.28 As
shown in Figure 1, all the fibrils are cylindrical nanofibrils, “Cubic 4” has 4 molecules
placed as a “cross” on each layer; “Cubic 5” has 5 molecules with the alanines radiating
outward; “Cubic 6” and “400 ns” has parallel placed Fmoc-AAs also with alanines radiating
outward. Except Cubic 4’s fibril, the plate layers are rotated 35.9 degrees relative to the
adjacent layer. The distance between each layer is 4.5 Å for SA and 400 ns models, and 5 Å
for all Cubic models. A lower density of molecules on each layer (like 2 or 3 Fmoc-AA
molecules on each layer) will result in an unstable fibril. Specific geometrical details are
listed in Table S1of supporting information.
All simulations and calculations were performed using GROMACS 4.5.4 and 4.5.329 with
OPLS-AA force field34,35, and applied periodic boxes with explicit water molecules. All
starting structures for MD simulation, except simulated annealing and replica exchange
molecular dynamics (REMD), were subjected to steepest descent energy minimizations with
5000 maximum steps to remove high-energy contacts. Before the productive MD, in order to
keep the system stable at 300 K and 1 bar, a 100 ps NVT and a 100 ps NPT equilibration
were applied respectively. During the productive MD, all bonds were constrained using
LINCS algorithm36,37; for every 10 fs, neighbors were searched in grid cells with 1 nm as
cutoff values for short-range neighborlist, electrostatic and van der Waals; long-range
electrostatics were treated with the particle mesh Ewald method38 with a grid spacing of
0.16 nm; constant temperature and pressure were maintained by coupling the system to an
external bath at 300 K and 1 bar, using velocity rescaling39 and Parrinello–Rahman40,
respectively. A 2 ps pressure coupling time was applied and the isothermal compressibility
of water is 4.5×10−5 bar−1. A leap-frog integrator with the integration time step of 2 fs was
used.41 GROMACS OPLS-AA force field with our fitted parameters for Fmoc group was
used for the Fmoc-AA, and a TIP3P model was applied for water.42 In SA of Fmoc-AA
molecules self-assembly, the initial fibril structure was the first 15 layers of the 400 ns
model. The system temperature was firstly heated up from 288 to 300 K in 0.5 ns, then
linearly and gradually cooled down to 0 K in the following 39.5 ns. We randomly extracted
120 frames during the temperature dropping from 700 to 290 K in SA that covered the
conformational space broadly, and those frames were then used in REMD as initial
structures; a set of temperatures ranging from 280 to 600 K was estimated and preset in
order to maintain the success rates of exchange within 20–30%.

[Assistant] [{"peptide_sequence": "AA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "3.3", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "35.0"}, {"peptide_sequence": "AA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "3.32", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "35.0"}, {"peptide_sequence": "AA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "3.34", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "35.0"}, {"peptide_sequence": "AA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "3.36", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "35.0"}, {"peptide_sequence": "AA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "3.38", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "35.0"}, {"peptide_sequence": "AA", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Fmoc", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "5.0", "PH": "3.4", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "35.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
The relationship between amino acid sequences and their resulting nanostructure
has been well studied, but that between amino acid chirality and nanostructure
handedness has not. Four dipeptide sodium salts with long alkyl chains derived
from L- and D-alanines were synthesized. The behavior of their self-assembly
into physical gels in water and THF was studied using field-emission scanning
electron microscopy, circular dichroism (CD), FT-IR spectroscopy, (1)H NMR
spectroscopy, and X-ray diffraction. The dipeptide salts organized into twisted
nanoribbons, whose handedness was controlled by the terminal alanine chirality.
The handedness of nanoribbons formed in water was opposite to that of those
formed in THF. The dipeptide salts self-assembled into similar interdigitated
bilayer structures in water and THF, but CD spectra of the gels indicated that
the stacking of carbonyl groups was opposite. The formation of this handedness
inversion is proposed to arise from the difference in interlayer distance and
chiral stacking of carbonyl groups near the C-terminals.

Materials. Stearic acid, methanol, sodium hydroxide, and alanines
were purchased from Sinopharm Chemical Reagent Co., Ltd.,
Shanghai, China.
Characterization of C15H31CO-L-Ala-L-Ala-ONa ((L,L)-2). FT-IR
(KBr, cm−1): 3300 (vN−H, amide A), 1634 (vCO, amide I), 1535
(vN−H, amide II). 1H NMR (400 MHz, DMSO-d6, TMS, 25 °C): δ =
0.85 (t, 3H; J = 7.0 Hz, CH3(CH2)12CH2), 1.12−1.23 (m, 30H;
CH3(CH2)12CH2, 2CH3CH), 1.48 (t, 2H; J = 6.8 Hz, CH2CH2CO),
J = 8.8 Hz, CH2CH2CO), 3.61−3.66 (m, 1H;
2.09 (t, 2H;
CONHCHCOONa), 4.17−4.21 (m, 1H; CONHCHCONH), 7.50−
7.54 (m, 1H, CONHCHCOONa), 8.10 (d, 1H,
J = 6.6 Hz,
CONHCHCONH). Elemental analysis for C15H31CO-L-Ala-L-Ala-
ONa·1/2H2O calcd (%): C, 61.51; H, 9.85; N, 6.52. Found: C, 61.21;
H, 10.35; N, 6.32.
Characterization of C15H31CO-L-Ala-D-Ala-ONa ((L,D)-2). FT-IR
(KBr, cm−1): 3300 (vN−H, amide A), 1634 (vCO, amide I), 1535
(vN−H, amide II). 1H NMR (400 MHz, DMSO-d6, TMS, 25 °C): δ =
0.85 (t, 3H; J = 6.6 Hz, CH3(CH2)12CH2), 1.12−1.24 (m, 30H;
CH3(CH2)12CH2, 2CH3CH), 1.48 (t, 2H; J = 7.0 Hz, CH2CH2CO),
J = 7.4 Hz, CH2CH2CO), 3.64−3.69 (m, 1H;
2.09 (t, 2H;
CONHCHCOONa), 4.16−4.22 (m, 1H; CONHCHCONH), 7.48−
J = 6.0 Hz,
7.52 (m, 1H, CONHCHCOONa), 8.09 (d, 1H,
CONHCHCONH). Elemental analysis for C15H31CO-L-Ala-D-Ala-
ONa·H2O calcd (%): C, 60.25; H, 9.88; N, 6.39. Found: C, 60.37; H,
10.27; N, 6.18.
Characterization of C15H31CO-D-Ala-L-Ala-ONa ((D,L)-2). FT-IR
(KBr, cm−1): 3300 (vN−H, amide A), 1635 (vCO, amide I), 1535
(vN−H, amide II). 1H NMR (400 MHz, DMSO-d6, TMS, 25 °C): δ =
0.85 (t, 3H; J = 6.6 Hz, CH3(CH2)12CH2), 1.12−1.23 (m, 30H;
CH3(CH2)12CH2, 2CH3CH), 1.48 (t, 2H; J = 6.4 Hz, CH2CH2CO),
J = 9.4 Hz, CH2CH2CO), 3.60−3.64 (m, 1H;
2.09 (t, 2H;
CONHCHCOONa), 4.16−4.20 (m, 1H; CONHCHCONH), 7.49−
7.54 (m, 1H, CONHCHCOONa), 8.10 (d, 1H,
J = 8.0 Hz,
CONHCHCONH). Elemental analysis for C15H31CO-D-Ala-L-Ala-
ONa·1/2H2O calcd (%): C, 61.51; H, 9.85; N, 6.52. Found: C, 61.22;
H, 9.87; N, 6.38.
Characterization of C15H31CO-D-Ala-D-Ala-ONa ((D,D)-2). FT-IR
(KBr, cm−1): 3300 (vN−H, amide A), 1634 (vCO, amide I), 1535
(vN−H, amide II). 1H NMR (400 MHz, DMSO-d6, TMS, 25 °C): δ =
0.85 (t, 3H; J = 7.0 Hz, CH3(CH2)12CH2), 1.12−1.23 (m, 30H;
CH3(CH2)12CH2, 2CH3CH), 1.48 (t, 2H; J = 6.8 Hz, CH2CH2CO),
J = 8.8 Hz, CH2CH2CO), 3.61−3.66 (m, 1H;
2.09 (t, 2H;
CONHCHCOONa), 4.17−4.21 (m, 1H; CONHCHCONH), 7.50−
7.54 (m, 1H, CONHCHCOONa), 8.10 (d, 1H,
J = 6.6 Hz,
CONHCHCONH). Elemental analysis for C15H31CO-D-Ala-D-Ala-
ONa·H2O calcd (%):C, 60.25; H, 9.88; N, 6.39. Found: C, 60.48; H,
10.49; N, 6.29.
Methods. FT-IR spectra of LMWGs were recorded from KBr
pellets of solids, on a Nicolet 6700 FT-IR spectrometer over the
4000−400 cm−1 range, with a resolution of 2.0 cm−1 and after
accumulation of 32 scans. FT-IR spectra of gels and solutions in D2O
were recorded from CaF2 windows, with a 50 μm spacer within a hot
stage. A D2O spectrum was used as the background and subtracted
from all sample spectra. FT-IR spectra of gels and solutions in THF-d8
were recorded from CaF2 windows, with a 50 μm spacer within a hot
stage. A THF-d8 spectrum was also recorded but not subtracted from
other spectra. 1H NMR spectra were recorded using a Varian NMR
400 spectrometer, in DMSO-d6 and THF-d8 solutions or gels using
tetramethylsilane (TMS) as an internal standard. Elemental analysis
was performed on a PerkinElmer series II CHNS/O analyzer 2400.
Field emission scanning electron microscopy (FESEM) images were
taken on a Hitachi S-4800 operating at 3.0 kV. Viscous aqueous
solutions were prepared at concentrations of 2.0 g L−1. FE-SEM
samples were prepared by dropping solutions on microscopy cover
glass. Samples were dried in air and then coated with platinum for 25 s
at 3.0 kV and 10 μA. THF organogel samples were prepared at
concentrations of 30 g L−1. FE-SEM samples were prepared by placing
a small amount of the gels on microscopy cover glass. Samples were
dried in air and then coated with platinum for 45 s at 3.0 kV and 10
μA. Small-angle X-ray diﬀraction (SAXRD) patterns were taken on an
X′Pert-Pro MPD X-ray diﬀractometer using Cu Kα radiation with a Ni
ﬁlter (1.542 Å). Hydrogels and organogels were prepared at
concentrations of 30 g L−1. Samples were prepared by drying the
gels on the surface of microscopy cover glass. The CD spectra were
obtained using an AVIV 410 spectrophotometer with a 1.0 nm
bandwidth, 1 scan, and an averaging time of 0.1 s at 25 °C using a 0.01
mm cell. Hydrogels and organogels were prepared at concentrations of
30 g L−1.

[Assistant] [{"peptide_sequence": "AA", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "aA", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "Aa", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "aa", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "2.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "AA", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 THF", "peptide_concentration(mg/ml)": "30.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "Aa", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 THF", "peptide_concentration(mg/ml)": "30.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "aA", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 THF", "peptide_concentration(mg/ml)": "30.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "aa", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 THF", "peptide_concentration(mg/ml)": "30.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "aa", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "10/90 THF/H2O", "peptide_concentration(mg/ml)": "30.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}, {"peptide_sequence": "aa", "self-assembly_phase": "ribbon", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "30/70 THF/H2O", "peptide_concentration(mg/ml)": "30.0", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "Yes", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
The core recognition motif of the amyloidogenic beta-amyloid polypeptide is a
dipeptide of phenylalanine. This dipeptide readily self-assembles to form
discrete, hollow nanotubes with high persistence lengths. The simplicity of the
nanotube formation, combined with ideal physical properties, make these
nanotubes highly desirable for a range of applications in bionanotechnology. To
fully realize the potential of such structures, it is first necessary to gain a
comprehensive understanding of their chemical and physical properties.
Previously, the thermal stability of these nanotubes has been investigated by
electron microscopy. Here, we further our understanding of the structural
stability of the nanotubes upon dry-heating using the atomic force microscope
(AFM), and for the first time identify their degradation product utilizing
time-of-flight secondary-ion mass spectrometry. We show that the nanotubes are
stable at temperatures up to 100 degrees C, but on heating to higher
temperatures begin to lose their structural integrity with an apparent collapse
in tubular structure. With further increases in temperature up to and above 150
degrees C, there is a degradation of the structure of the nanotubes through the
release of phenylalanine building blocks. The breakdown of structure is observed
in samples that are either imaged at elevated temperatures or imaged following
cooling, suggesting that once phenylalanine is lost from the nanotubes they are
susceptible to mechanical deformation by the imaging AFM probe. This
temperature-induced plasticity may provide novel properties for these peptide
nanotubes, including possible applications as scaffolds and drug delivery
devices.

Preparation of Peptide Samples. FF peptides were purchased from
Bachem (Bubendorf, Switzerland). Fresh stock solutions were prepared
by dissolving the lyophilized peptides in 1,1,1,3,3,3-hexafluoro-2-
propanol (Sigma Aldrich) at a concentration of 100 mg/mL. Peptide
stock solution was diluted in double distilled (dd) H2O to a final
concentration of 2 mg/mL. To prevent overcrowding of the scan area,
samples were diluted in Elgar water to a final concentration of 0.2 or

[Assistant] [{"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "1/99 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "5.5", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "1/99 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "5.5", "temperature(celsius)": "50.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "tube", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "1/99 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "5.5", "temperature(celsius)": "100.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "1/99 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "5.5", "temperature(celsius)": "150.0", "heating_cooling": "No", "incubation_period(min)": "nan"}, {"peptide_sequence": "FF", "self-assembly_phase": "none", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "1/99 HFP/H2O", "peptide_concentration(mg/ml)": "1.0", "PH": "5.5", "temperature(celsius)": "200.0", "heating_cooling": "No", "incubation_period(min)": "nan"}]


[System] polypeptide self-assembly literature information extraction

[User]
Peptides that self-assemble into nanostructures are of tremendous interest for
biological, medical, photonic and nanotechnological applications. The enormous
sequence space that is available from 20 amino acids probably harbours many
interesting candidates, but it is currently not possible to predict
supramolecular behaviour from sequence alone. Here, we demonstrate computational
tools to screen for the aqueous self-assembly propensity in all of the 8,000
possible tripeptides and evaluate these by comparison with known examples. We
applied filters to select for candidates that simultaneously optimize the
apparently contradicting requirements of aggregation propensity and
hydrophilicity, which resulted in a set of design rules for self-assembling
sequences. A number of peptides were subsequently synthesized and characterized,
including the first reported tripeptides that are able to form a hydrogel at
neutral pH. These tools, which enable the peptide sequence space to be searched
for supramolecular properties, enable minimalistic peptide nanotechnology to
deliver on its promise.

9. Marchesan, S., Easton, C. D., Kushkaki, F., Waddington, L. & Hartley, P. G.
Tripeptide self-assembled hydrogels: unexpected twists of chirality. Chem.
Commun. 48, 2195–2197 (2012).
11. Marchesan, S. et al. Chirality effects at each amino acid position on tripeptide
self-assembly into hydrogel biomaterials. Nanoscale 6, 5172–5180 (2014).
12. James, J. & Mandal, A. B. The aggregation of Tyr-Phe dipeptide and Val-Tyr-Val
tripeptide in aqueous solution and in the presence of SDS and PEO–PPO–PEO
triblock copolymer: ﬂuorescence spectroscopic studies. J. Colloid Interface Sci.
360, 600–605 (2011).
13. Reches, M., Porat, Y. & Gazit, E. Amyloid ﬁbril formation by pentapeptide
and tetrapeptide fragments of human calcitonin. J. Biol. Chem. 277,
35475–35480 (2002).
14. Hauser, C. A. E. et al. Natural tri- to hexapeptides self-assemble in water to
amyloid beta-type ﬁber aggregates by unexpected alpha-helical intermediate
structures. Proc. Natl Acad. Sci. USA 108, 1361–1366 (2011).
where the prime indicates the normalization of the respective variable between 0 and
1. As logP(trip) is a unitless number linearly proportional to ΔGwater-oct and is
normalized in equation (1), it was chosen to deﬁne it simply as the sum of the
Wimley–White whole-residue hydrophobicities39,40 ΔGwater-oct (kcal mol
tripeptide, given by
Preparation of tripeptide samples. Tripeptides were dissolved in water by
sonication and vortexing, followed by bringing the pH to 7.2 ± 0.1 by dropwise
addition of 0.25 M NaOH solution to a ﬁnal concentration of 30 mmol l
−1 for DLS and NMR experiments. Note that the
These were diluted to 10 mmol l
KYF gel could also be prepared by direct dissolution into a 0.1 M phosphate buffer
at pH 7. Characterization of the resulting samples took place at least 24 h after
sample preparation. Samples for FTIR and NMR analysis were prepared using
D2O (99.9% D) and NaOD solution instead of H2O and NaOH.

[Assistant] [{"peptide_sequence": "KYF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "13.7", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KYY", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "14.2", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KFF", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "13.2", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}, {"peptide_sequence": "KYW", "self-assembly_phase": "hydrogel", "N-terminal_modification": "Free", "C-terminal_modification": "Free", "Non-terminal_modification": "nan", "peptide_conjugate_mixture": "Peptide", "conjugate_partner": "nan", "solution": "100 H2O", "peptide_concentration(mg/ml)": "14.9", "PH": "7.0", "temperature(celsius)": "25.0", "heating_cooling": "No", "incubation_period(min)": "1440.0"}]


[System] polypeptide self-assembly literature information extraction

[User]
igin the life.
In order to examine the morphology of nanostructures formed by the self-assembling peptides listed
in  Table  1,  structural  evaluation  was  performed    using  low-temperature  FESEM.  Previously,  we  had
reported  a  critical  gelation  concentration  for  the  peptides  to  form  hydrogels  [13].  Nonetheless,  we
observed hollow nanospheres and structures resembling membrane blebs already at concentrations far
below  that  required  for  gelation  (Figure  1).  For  ID3,  membrane  structures  and  hollow  nanospheres
were  observed  at  concentrations  of  2  and  5  mg/mL  respectively,  far  below  the  critical  gelation
concentration  of  10  mg/mL  (Figure  1A,B).  At this  concentration,  no  gelation  or  fiber  networks  were
seen. For LD6, hollow  nanospheres and  membranous  blebs were  seen  at a  low peptide concentration  of
0.1 mg/mL (Figure 1C,D), around 10 times lower than that required for gelation. Membrane structures
were  also  observed  for  LD6  and  AD6  at  concentrations  near  the  critical  gelation  concentration  (Figure
1F).  For  AD6,  hollow  nanospheres  were  seen  at 5  mg/mL,  close  to the  critical  concentration  (Figure
1E). The hollow nanospheres (Figure 1E) seem to be composed of condensed fibers. The factors that
drive  further  organization  of  assembled  fibers  into  nanospheres  and  meshes  are  not  yet  clear.  It  is
likely  that  the  process  is  controlled  by  specific  thermodynamic  parameters.  The  nanospheres  can  be
considered equivalent to the hydrogel cavities that are formed at higher concentrations. The nanospheres
formed by these amphiphilic self-assembling peptides at low concentrations have potential applications
as natural vehicles or carriers for active and passive drug delivery.
The first formulation of a theory on a strictly materialistic basis for the origin of life was Haldane’s
and  Oparin’s  hypothesis  of  a  primordial  soup,  containing  all  essential  chemicals  required  to  form
biomolecules  of  increasing  complexity  by  a  process  of  chemical  evolution  [15].  Miller  demonstrated
that amino acids can be formed in a model atmosphere containing methane, hydrogen, ammonia, water
and some other simple compounds under the influence of electric discharges and heat [16]. Miller’s work
laid  the  foundation  for  exploring  pre-biotic  chemistry  by  using  controlled  model  environments  [17].
Many  of  these  gas-phase  experiments  as  reviewed  by  Rode  et  al.  point  to  the  existence  of  simple,
aliphatic  amino  acids  such  as  leucine  (L),  glycine  (G),  valine  (V),  alanine  (A),  aspartic  acid  (D)  and
glutamic acid (E) in the primordial soup [17,18]. The Salt Induced Peptide Formation (SIPF) reaction
in  combination  with  adsorption  processes  on  clay  minerals  provides  one  of  the  simplest  and  most
universal mechanisms for production of the first peptides on Earth [17]. After SIPF reaction times of a
few days, heptapeptides were the largest sub-units analyzed [19]. This makes our study of self-assembling
ultrasmall  peptides,  made  of  simple  aliphatic  residues,  such  as  LIVAGD,  AIVAGD  and  IVD,
especially  important  in  the  context  of  better  understanding  prebiotic  chemical  evolution.  Hollow
spheres  and  membrane  blebs  formed  by  these  amphiphilic  ultrasmall  peptides  (Figure  1A,C–E)  can
have diameters between a few hundred nanometers to a micrometer. These self-assembled supramolecular
structures  may  have  very  likely  played  an  important  role  in  biological  evolution  and  speciation  by
providing  specialized  niches or microenvironments. The acidic pH and  high salt concentration of the
primordial  soup  may  have  caused  immediate  degradation  of  oligonucleotides  and  hydrolysis  of
sensitive  compounds  [17].  In  this  regard,  self-assembled  peptide  nanostructures  that  are  more  stable
Figure  1.  Morphological  characterization  of  the  supramolecular  structures  formed  by
aliphatic  ultrasmall  peptides  at  low  concentrations  (close  to  or  far  below  the  minimum
gelation  concentration)  using  FESEM.  Representative  images  of  hollow  nanospheres  and
blebs formed by (A) Ac-ID3 (L) (5 mg/mL) at 8000×; (B) Ac-ID3 (L) (2 mg/mL) at 2200×;
(C,  D)  Ac-LD6  (L)  (0.1  mg/mL)  at  4000×  and  15,000×  respectively;  (E)  Ac-AD6  (L)  (5
mg/mL) at 37,000×; (F) Ac-LD6 (L) (1.3 mg/mL) at 6000×.
Our unique class of ultrasmall aliphatic peptides forms hydrogels when dissolved in water at higher
concentrations. The opacity and mechanical stiffness of the hydrogels increase with increasing peptide
concentration  [13].  Near  the  critical  gelation  concentration,  clear  gels  were  formed  within  hours
(Figure  2B,C).  At  higher  concentrations,  gelation  occured  within  minutes,  giving  translucent  white
gels with higher  mechanical  stiffness (Figure 2A). The hydrogels were shock  frozen, lyophilized and
observed under FESEM. Dense, interconnected networks of long, twisted helical fibers were observed
(Figure  3).  These  long  fibers  entangle  together  to  create  meshes  that  can  entrap  water  and  form
hydrogels. Majority of water molecules surround the peptide  fibers (hydrophilic exterior) and are not
retained  within  them  (hydrophobic  core)  [12].  These  supramolecular  assemblies  can  be  considered
β-turn  rather  than  β-sheet  structures  due  to  the  helical  turns  and  twists  of  the  fibers.  This  was
confirmed  by  CD  spectroscopy  and  X-ray  fiber  diffraction  [12].  Each  helical  turn  in  an  α-helix
corresponds to 3.6 amino acids and defined α-helices are formed in water by cyclic pentapeptides [20].
Thus,  there  is  a  general  view  that  linear  tri-  to  heptapeptides  are  restricted  by  size  to  form  helical
structures  in  aqueous  solutions.  In  this  context,  it  is  interesting  that  even  the  trimers  aggregate  into
fibers with helical twists and turns. We want to emphasize that our self-assembling peptides were not
specifically designed to form α-helices. For instance, this  is unlike the 28-residue peptides developed
by Woolfson et al. with a coiled-coil heptad sequence repeat that form hydrogels through longitudinal
stacking of α-helical dimers [21]. The self-assembly of our peptides  involves distinct conformational
transitions from random coil to anti-parallel, helical dimer pairs, which then stack on top of each other
to  form  fibers  [12].  The  emergence  of  these  supramolecular  structures  relies  on  the  efficiency  of
conformational transitions, which are in turn dictated by the amino acid sequence.
Recent  studies  on  the  physical  properties  of  hydrogels  formed  by  these  aliphatic  peptides
demonstrated their temperature-resistance and high mechanical stiffness that can be tuned by changing
the  pH,  salt  and  peptide  concentration  [13].  In  addition,  the  chemical  composition  can  be  modified
through surface functionalization for conjugation of growth factors, matrix metalloproteinase cleavable
sequences  and  cell  adhesion  factors  [22,23].  The  biocompatibility  of  the  peptide  hydrogels  with
different types of cells has also been demonstrated [13]. Thus, the peptide hydrogels can be used to make
functional 3-D scaffolds for tissue engineering and injectable formulations for regenerative medicine.
Figure  2.  Representative  pictures  of  hydrogels  formed  by  self-assembling  peptides
containing aliphatic amino acids. Inverted vials show the formation of solid, stable hydrogels
(A) Ac-ID3 (L) 14 mg/mL; (B) Ac-AD6 (L) 5 mg/mL; (C) Ac-LD6 (L) 2.5 mg/mL.
Figure  3.  Morphological  characterization  of  the  supramolecular  networks  formed  by
aliphatic  ultrasmall  peptides  using  FESEM.  Representative  images  of  fibers  formed  by
(A, B) Ac-ID3 (L) (15 mg/mL) at 30,000× and 100,000× respectively; (C, D) Ac-AD6 (L)
(5  mg/mL)  at 30,000×  and 95,000× respectively;  (E)  Ac-LD6 (L) (10  mg/mL)  at  35,000×;
(F) Ac-LD6 (L) (2 mg/mL) at 150,000×.
Design  of  the  peptide  sequence  holds  the  cues  required  for  non-covalent  interactions  that  drive
molecular  recognition  and  self-assembly.  Hence,  it  is  necessary  to  explore  the  effect  of  sequence
modification on self-assembly  and supramolecular organization of this unique class of peptides. This
was  done  by  replacing  the  aspartic  acid  residue  of  LD6  and  ID3  at  the  polar  C-terminus  with  an
aromatic  amino  acid  such  as  tyrosine,  phenylalanine,  tryptophan.  This  modification  had  a  marked
influence  on  the  ease  of  self-assembly,  the  gelation  capacity  and  the  morphology  of  the  fibers
(Figure  4,5).  Of  the  six  different  sequences  with  aromatic  residues,  only  those  with  phenylalanine
formed  relatively  stable  hydrogels  (Figure  4A,B).  IW3  formed  a  weak  hydrogel  that  collapsed  into
smaller  aggregates  in  aqueous  solution  within  72  h  of  formation  (Figure  4F).  The  remaining  three
sequences, namely IY3, LY6 and LW6 only formed small aggregates in solution (Figure 4C–E). These
results can be attributed to the formation of short, straight fibers by the  modified peptides (Figure 5).
The short and flat fibers are unable to entangle and make dense meshes required to effectively entrap
water to produce stable hydrogels. This is due to their inability to bend and curl. Some peptides formed
straight  fibers  with  a  flat  morphology  (Figure  5A–D).  These  flat  nanostructures  are  similar  to
assemblies observed  for fully aromatic di-tripeptides such as FF and FFF [24]. These assemblies can
be regarded as flat -sheets rather than -turns observed for the aliphatic sequences.
Figure  4.  Representative  pictures  of  hydrogels/aggregates  formed  by  self-assembling
peptides  containing  an  aromatic  amino  acid  at  the  C-terminus.  Inverted  vials  show  the
formation of solid, stable hydrogels. (A) Ac-LF6 (L) 10 mg/mL; (B) Ac-IF3 (L) 5 mg/mL;
(C)  Ac-LY6  (L)  10  mg/mL;  (D)  Ac-IY3  (L)  5  mg/mL;  (E)  Ac-LW6  (L)  10  mg/mL;
(F) Ac-IW3 (L) 5 mg/mL after 72 h.
Figure  5.  Morphological  characterization  of  the  supramolecular  networks  formed  by
ultrasmall  peptides  containing  an  aromatic  amino  acid  as  head  group  using  FESEM.
Representative images of fibers formed by (A, B) Ac-LW6 (L) (10 mg/mL) at 40,000× and
100,000× respectively; (C, D) Ac-IY3 (L) (10 mg/mL) at 25,000× and 80,000× respectively;
(E, F) Ac-IF3 (L) (5 mg/mL) at 80,000× and 100,000× respectively.
The  higher  stability  of  hydrogels  formed  by  phenylalanine-containing  peptides  compared  to  the
other modified sequences can be explained by considering the chemical structure of different aromatic
amino acid residues. The lone pair of electrons on the nitrogen (in tryptophan) and oxygen (in tyrosine)
can interact with the π-electrons of the aromatic ring, giving resonance structures. The same cannot be
said  for  the  phenylalanine  residue,  which  cannot  form  resonance  structures.  Also,  IF3  forms  more
rounded,  cylindrical  fibers  compared  to the  flat  fibers  formed  by  the  other  sequences  (Figure  5E–F).
The  round  fibers  probably  form  a  better  mesh  compared  to  the  flat  fibers.  In  addition,  a  cylindrical
fiber  has  much  greater  total  surface  area  than  a  cuboidal  fiber  of  comparable  dimensions.  This
increases  the  water  retention  capacity  for  the  cylindrical  fibers  as  a  greater  area  of  the  hydrophilic
exterior is in contact with the water molecules.
The  occurrence  of  aromatic  amino  acids  in  naturally  occurring  motifs  such  as  amyloidogenic
sequences  has  led  to the  general  view  that these  residues  are  essential  for  molecular  recognition  and
self-assembly  [25].  The  exact  mechanism  for  aromatic  stabilization  in  self-assembly  is  unclear  and
proposed theories  include π-stacking or hydrophobic  interactions among aromatic residues. T-shaped
stacking  of  the  aromatic  rings,  with  the  planar  surfaces  roughly  perpendicular  to  each  other  has  also
been reported [24,26,27]. Furthermore, it has been suggested that aromatic amino acids are important
for  self-assembly  due  to  their
increased  hydrophobicity  and  β-sheet  propensity  rather  their
aromaticity  [28].  Compared  to  our  aliphatic  peptides,  the  modified  aromatic  sequences  form  straight
fibers without helical turns or twists. The introduction of a planar aromatic ring at the C-terminal head
introduces a certain degree of steric hindrance that prevents the formation of helical turns in the fibers.
This steric effect is a possible explanation for the lack of long, extended assemblies for these modified
peptides. Furthermore, crystallographic work done by Gorbitz et al. on the FF peptide suggests that the
assembled  nanotubes  may  be  structurally  similar  to  FF  crystals  [26].  On  the other  hand,  Gazit  et  al.
concluded  that  the  crystal  packing  of  the  peptide  represented  a  completely  different  molecular
arrangement  compared  to  the  individual  self-assembled  nanotubes  [29].  More  investigations  clearly
need to be conducted in order to determine the exact mechanism  by which aromatic residues  interact
and influence self-assembly.
The  short,  straight  nanostructures  produced  by  self-assembly  of  modified  peptide  sequences  can
provide  the  building  blocks  for  development of  modern  nanodevices.  For  example,  Gazit  et  al.  have
demonstrated  the  use  of  L-diphenylalanine  peptide  nanotubes  as  degradable  molds  for  casting  silver
nanowires  [29].  These  nanotubes  have  also  been  deposited  on  the  surface  of  simple  screen-printed
electrodes,  significantly  enhancing  their  sensitivity  [30].  The  remarkable  improvement  caused  by
nanotube  deposition  was  linked  to

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