[System] polypeptide self-assembly literature information extraction

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Tubular nanostructures are suggested to have a wide range of applications in
nanotechnology. We report our observation of the self-assembly of a very short
peptide, the Alzheimer's beta-amyloid diphenylalanine structural motif, into
discrete and stiff nanotubes. Reduction of ionic silver within the nanotubes,
followed by enzymatic degradation of the peptide backbone, resulted in the
production of discrete nanowires with a long persistence length. The same
dipeptide building block, made of D-phenylalanine, resulted in the production of
enzymatically stable nanotubes.

Casting Metal Nanowires Within
Discrete Self-Assembled Peptide
Nanotubes
Meital Reches and Ehud Gazit*
Tubular nanostructures are suggested to have a wide range of applications in
nanotechnology. We report our observation of the self-assembly of a very short
peptide, the Alzheimer’s (cid:2)-amyloid diphenylalanine structural motif, into dis-
crete and stiff nanotubes. Reduction of ionic silver within the nanotubes,
followed by enzymatic degradation of the peptide backbone, resulted in the
production of discrete nanowires with a long persistence length. The same
dipeptide building block, made of D-phenylalanine, resulted in the production
of enzymatically stable nanotubes.
Self-assembled nanostructures provide a key
direction for the controlled fabrication of novel
nanoscopic materials and devices. Nanotubular
structures are particularly important structural
elements because they may serve as nanowires
or nanoscaffolds (1–4). Peptide nanotubes are
especially intriguing assemblies because they
have the scope for numerous chemical modifi-
cations and allow the use of biological systems
specificity. A milestone in the production of
peptide-based nanotubes was the demonstra-
tion by Ghadiri et al. (2, 5) that a cyclic
octapeptide with alternating L and D amino
acids can form a nanotubular structure by
self-assembly. However, the peptide nano-
tubes that formed further assembled into an
array of tubes that were aligned as crystalline
microscopic structures (2, 5). Linear surfac-
tant-like hepta- and octapeptides can also
self-assemble into a network of open-ended
from which nanovesicles can
nanotubes,
“bud” or “fuse” (3, 4). Peptide-related bis(N-
(cid:3)-amido-glycylglycine)-1,7-heptane
boxylate molecules were also shown to be
assembled into tubular structures (6).
We observed the formation of peptide
nanotubes while studying the ability of very
short aromatic peptides (hexapeptides and
shorter) to form well-ordered amyloid fibrils
(7–9). Amyloid fibrils are the hallmark of a
diverse group of diseases of unrelated origin,
including Alzheimer’s disease, type II diabe-
tes, and prion diseases (10–15). Despite their
formation by a diverse and structurally unre-
lated group of proteins, all amyloid fibrils
share similar biophysical and structural prop-
erties. On the basis of both experiments and
theory, we recently suggested that stacking of
aromatic residues may play a key role in the
process of molecular recognition and self-
assembly that leads to the formation of amy-
loid fibrils (16). This proposal is in agree-
ment with the well-known role of aromatic
stacking in the formation of chemical and
biochemical supramolecular structures (17–
19). According to our suggestion, the restrict-
ed geometry and the attractive forces of the
aromatic moieties provide order and direc-
tionality as well as the energetic contribution
needed for the formation of such well-or-
dered structures. In this context, we were
intrigued to determine the molecular proper-
ties of a peptide fragment corresponding to
the core recognition motif of A(cid:2), the diphe-
nylalanine element (Fig. 1A). This motif is of
special interest, because several studies have
identified the ability of larger peptides and
conjugated organic molecules that contain
this motif to inhibit fibril formation by A(cid:2)
(20–22). Some of those inhibitors are current-
ly undergoing clinical trials as potential drugs
to treat Alzheimer’s disease (23, 24).
Here, we sought to make the NH2-Phe-
Phe-COOH dipeptide soluble at very high
concentrations ((cid:1)100 mg/ml) by dissolving
1,1,1,3,3,3
hexafluoro-2-propanol. Although the peptide
appeared to be highly soluble in the organic
solvent, a rapid assembly into ordered semi-
crystalline structures was observed visually
within seconds after dilution into the aqueous
solution at a final (cid:4)M concentration range.
We used dynamic light scattering analysis to
assembly into supramolecular
determine
structures within minutes at the (cid:4)M range.
Transmission electron microscopy (TEM)
analysis with negative staining indicated that
the peptide forms well-ordered, tubular, and
elongated assemblies (Fig. 1B). The light
shell and the dark center, as observed in Fig.
1B, suggested hollow tubular structures filled
with the negative stain, uranyl acetate. Ener-
gy-dispersive x-ray analysis (EDX) indicated
the presence of uranium within the assembled
structures
structures
(fig. S1A). Smaller
could also be observed by TEM analysis; we
propose that these represent smaller tubular
assemblies or fragments of the larger nano-
tubes. The persistence length of the nano-
tubes appears to be on the order of microme-
ters, as evident by the microscopic observa-
tion. The formation of the tubular structures
was very efficient. Almost all the assemblies
as observed by TEM had tubular structures,
and almost no amorphous aggregates were
observed ((cid:5)1%). This observation is in
marked contrast to other peptide assemblies
(such as amyloid fibrils) in which a mixture
of ordered and aggregated structures may be
observed. High-resolution field emission gun
TEM (HR-TEM) (Fig. 1C) provided further
indication of the regular structures of the tube
walls. The formed structures were highly or-
dered and appeared to be rather stiff but
without the usual branching and curving typ-
ical of amyloid fibrils. On the other hand, the
assemblies show some morphological simi-
larity in terms of size and tubular structures to
the recently observed peptide nanotubes that
are formed by much longer surfactant-like
hepta- to octapeptides (3). These structures
are different from the first-reported peptide
nanotubes
formed by cyclic
polypeptides made of alternating D and L
amino acids (5).
We used scanning electron microscopy
(SEM) to further study the tubular structures
(Fig. 2, A and B). The nanotubes were applied
on a glass coverslip coated with gold and im-
aged by SEM. The low-magnification micro-
graphs of areas filled with individual nanotubes
(Fig. 2A) further demonstrate that the tubes are
relatively homogeneous and evidently are indi-
vidual entities with a persistence length on the
order of micrometers. Figure 2C shows the
statistical distribution of the diameters of the
nanotubes. The crystal structure of the diphe-
nylalanine peptide, as formed by evaporation of
aqueous solution at 80°C, showed a crystal
packing of aligned and elongated long hollows
Fig. 1. Self-assembly of well-ordered and elongated peptide nanotubes by a molecular recognition
motif derived from the (cid:2)-amyloid polypeptide. (A) The central aromatic core of the (cid:2)-amyloid
polypeptide is involved in the molecular recognition process that leads to the formation of amyloid
ﬁbrils. Various fragments of the core form amyloid ﬁbrils or inhibit their formation. (B) TEM images
of the negatively stained nanotubes formed by the diphenylalanine peptide. (C) HR-TEM images of
negatively stained peptide nanotubes, visualized by ﬁeld emission gun microscope.
Fig. 2. Structural anal-
the tubular
nanoparticles. (A) Low-
magniﬁcation SEM im-
ages of a ﬁeld of dis-
crete nanotubes that
are present as individual
entities. (B) High-mag-
niﬁcation SEM image of
an individual nanotube.
(C) A statistical dis-
tribution of nanotube
diameters.
(25). These structures are also referred to as
peptide nanotubes. However, our structural
analysis indicates that the crystal packing of the
peptide represents a completely different mo-
lecular arrangement compared with the self-
structures.
assembled
Higher magnification SEM analysis also in-
dicated a typical nanotubular structure that
resembled, to some extent, a class of peptide
nanotubes that were recently reported (3),
although our structures are apparently stiffer
and discrete (Fig. 2B).
To gain insight into the molecular con-
figuration of the assembled structures, we
used Fourier-transformed infrared spectros-
copy (fig. S2). Spectral analysis of the
assemblies showed a sharp 1630 cm(cid:6)1 peak
at the amide I region. This peak is consis-
tent with a (cid:2) sheet–like conformation of the
single amide bond, as was suggested for
peptide nanotubes built of larger building
blocks (1– 4 ) and for amyloid fibrils (9).
Because the dipeptide contains only a sin-
gle amide bond, the vibrational assignment
of this peak is definitive and does not rep-
resent an averaged contribution of several
bonds, as occurs when longer peptide as-
semblies are analyzed. Moreover, no other
vibrational peaks are observed (such as ran-
dom coil peak around 1654 cm(cid:6)1) (26 ),
which further suggests a uniform confor-
mation of all the building blocks.
The formation of peptide nanotubes by
the dipeptide is very interesting when
viewed in the context of the mechanism of
amyloid formation. Max Perutz recently
suggested that amyloid fibers are water-
filled nanotubes (27 ). The tubular struc-
tures formed by the dipeptides show a
green-gold birefringence upon staining
with Congo red dye, which is consistent
with an organization that may be similar to
that of amyloid structures. Therefore,
indicates that the extremely small core rec-
ognition motif of the (cid:2)-amyloid polypeptide
contains all the molecular information needed
to mediate self-assembly into regular struc-
tures. The context in which the diphenylala-
nine recognition motif is set apparently dic-
tates its assembly either into canonical amy-
loid fibrils, as is the case of A(cid:2), or into stiff
nanotubes, as described here.
Downloaded from https://www.science.org at Massachusetts Institute of Technology on March 21, 2024To study whether the tubes are indeed
hollow and filled with aqueous solution, we
added ionic silver to the nanotubes in solu-
tion. HR-TEM visualization followed by
EDX analysis indicated that silver nanopar-
ticles were formed within the tubes (fig.
S1B). On the basis of these observations,
we examined the ability of the nanotubes to
serve as molds for casting metal nanowires
(Fig. 3A). The tubes were added to boiling
ionic silver solution, and the silver was
reduced with citric acid to ensure a more
uniform assembly of the silver nanowires
(28, 29). TEM analysis (without staining)
indicated the formation of metal assemblies
within the majority (80 to 90%) of the tubes
(Fig. 3B). Proteolytic lysis of the peptide
mold, by the addition of a proteinase K
enzyme to the silver-filled nanotubes solu-
tion, resulted in the attainment of individ-
ual silver nanowires (cid:7)20 nm in diameter as
seen by TEM (Fig. 3, C and D). The diam-
eter of the nanowires is smaller than that
of the tubes, which further suggests that
casting was done inside the tubular struc-
identity of the wire
ture. The chemical
was confirmed by EDX analysis (fig. S1C).
For other applications, such as the as-
sembly of nanotube-based biosensors or
hollow tubing of nanofluidic circuits, enzy-
matically stable nanotubes are desirable. To
assemble such stable tubes, we used pro-
teolitically stable building blocks based on
the D–amino acid analog of the peptide,
NH2-D-Phe-D-Phe-COOH. This
formed nanotubes with the same structural
features as the corresponding L–amino acid
peptide (Fig. 4A). After 1 hour of incubation
of the Phe-Phe peptide with proteinase K
(0.02 mg/ml), no tubular structures were ob-
served by electron microscopy examination
(as compared with hundreds of tubular struc-
tures observed before the proteolysis). In
marked contrast, no notable variation could
be observed before and after the incubation of
the D-Phe-D-Phe peptide with the enzyme.
In light of the formation of nanotubes by
such short dipeptides, we tested the ability of
other aromatic dipeptides (Phe-Trp, Trp-Tyr,
Trp-Phe, and Trp-Trp) under similar condi-
tions. Nanoscale tubular structures were ob-
served only in the case of the Phe-Trp peptide
Fig. 3. Casting of silver nanowires with the peptide nanotubes. (A) The nanowires were formed by
the reduction of silver ions within the tubes, followed by enzymatic degradation of the peptide
mold. (B) TEM analysis (without staining) of peptide tubes ﬁlled with silver nanowires. (C and D)
TEM images of silver nanowires that were obtained after the addition of the proteinase K enzyme
to the nanotube solution.
Fig. 4. Formation of peptide
nanotubes by different aromatic
peptides. (A) TEM image of the
stable nanotubes formed by the
D–amino acid building-block an-
alog (B) TEM image of a tubular
structure formed by the NH2-
Phe-Trp-COOH dipeptide. Amor-
phous aggregates can be ob-
served in the background.
Our studies present the synthesis of pep-
tide nanotubes that are separated, are ho-
mogeneous, and have a long persistence
length. The unique properties of the tubes
and their proteolytic lability allow their use
as nanoscale molds for the casting of silver
nanowires. For other applications, enzy-
matically stable tubes can be fabricated.
15. R. B. Wickner et al., J. Struct. Biol. 130, 310 (2000).
16. E. Gazit, FASEB J. 16, 77 (2002).
17. M. L. Waters, Curr. Opin. Chem. Biol. 6, 736 (2002).
18. A. S. Shetty, J. Zhang, J. S. Moore, J. Am. Chem. Soc.

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[System] polypeptide self-assembly literature information extraction

[User]
Copyright © 2013 Elsevier Inc. All rights reserved.

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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