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Self-assembly of aromatic amino acids: a

molecular dynamics study†
Cite this: Phys. Chem. Chem. Phys.,
2018, 20, 30525 a
Sahin Uyaver, Helen W. Hernandez *b and M. Gokhan Habiboglua

Received 7th October 2018,
Accepted 27th November 2018
DOI: 10.1039/c8cp06239k
rsc.li/pccp
The self assembly processes of aromatic amino acids, phenylalanine, tyrosine, and tryptophan have been
simulated and were observed to form fibril-like aggregates linked to certain rare diseases and instances
of biological membrane disruption. Pure systems and their mixtures were studied systematically at
constant temperatures and free energy landscapes were produced describing the height and the
number of assembled monomers associated with lower energy structures. Consistent with some
previous work, aromatic amino acid monomers display a tendency to arrange with a four-fold
symmetry. The occurrence of this and other ordered structures increases at higher temperatures.
At lower temperatures our binary mixture simulations indicate that increasing tryptophan content drives
the assembly process away from the formation of distinct nanostructures and toward disordered
aggregates which is in line with experimental observations of pure tryptophan solutions. This work
provides molecular level insight to a variety of different physical phenomena relevant to fields including
human disease.

Introduction
Amino acids are used as biological building blocks for proteins
and they are involved in complex metabolic cycles in the body
under normal conditions. In some instances, the body is not
able to correctly metabolize an amino acid, and the conse-
quences can be quite severe. When this occurs with an aromatic
amino acid, phenylalanine (PHE), the result is a disease called
phenylketonuria (PKU).1–3 Phenylketonuria is a rare disease
occurring in less than 20 000 individuals in the US. Like many
rare diseases, there is a distinct lack of economic incentive to
perform fundamental research and/or embark on expensive
drug discovery efforts that would result in a treatment with a
very limited market. If left untreated, this disease progresses
until the individual has severe mental retardation and usually
demands intensive care. For a long time the source of this
mental deterioration was unknown and assumed to be asso-
ciated with excess phenylalanine molecules competing for
passage through the blood brain barrier and preventing other
compounds from doing so in the required amounts. In 2009,
Adler-Abramovich et al. demonstrated that individual phenyla-
lanine molecules assemble into fibrils and that those fibrils
exhibit cellular toxicity and are present in the brains of mice
and humans with PKU. In addition, they showed that PHE
a
Turkish-German University, Sahinkaya Cad, 86, 34820, Beykoz, İstanbul, Turkey
b
KAL Research Initiatives LLC, Houston, Texas 77042, USA.
E-mail: helenwhernandez@kalresearchinitiatives.com
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cp06239k
fibrils (both synthesized and isolated from mice with PKU)
exhibit the histological characteristics of amyloid plaques.4
This work has led to a new paradigm of understanding of
PKU and has spurred on a considerable amount of related
research for this rare disease and others.
In our previous simulation work, we were the first to observe
the formation of individual phenylalanine molecules into a
tubular structure with a four-fold symmetry by using the OPLS
(Optimized Potentials for Liquid Simulation) force field.5 Our
simulation result showed a four-fold tube in which hydrophilic
termini were internal to the structure with hydrophobic rings
external exposed to water. As noted in a recent review,6 the
structure we described was initially quite surprising as typically
the hydrophobic regions would be expected to be buried to
reduce exposure to water molecules. However, the existence
of these assemblies has been confirmed by Do et al. who
used ion mobility electrospray ionization mass spectrometry
(IM-ESI-MS) to observe both the mass to charge ratios and the
cross sectional areas of phenylalanine oligomers. These values
aligned very well with our model and provides experimental
support that phenylalanine molecules form fibrils with layers
of four. They also consider fibrillar growth and suggest that
the fibrils prefer to grow alongside each other with close
association of aromatic rings reminiscent of the steric zipper
classically observed in amyloid fibrils.7
In recent years, much has been discovered about phenyl-
alanine self-assembly, but less is known about the other two
aromatic amino acids. Tyrosine (TYR) has been observed to
form very straight fibrils on a variety of different substrates8,9

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An inability to break down tyrosine is associated with tyrosinemia
recently suggested to fall into the category of amyloid-like
metabolic diseases.10 In alkaptonuria, the build-up of meta-
bolite of a tyrosine, homogentisic acid,11–13 also occurs in
typical amyloidogenic manner as evidenced by interaction with
Congo Red dye.10,13
Very little work has been performed to understand trypto-
phan (TRP) self-assembly and to our knowledge, no simulation
work on this process has been performed. Due to its rare status,
research on hypertryptophanemia is extremely limited but it
has been described previously in relation to tryptophan struc-
ture formation.14 Additionally, tryptophan has been shown to
disrupt certain biological membranes. In fact, tryptophan
disrupted the thylakoid membranes at lower concentrations
and to a much greater extent than did phenylalanine.15 The
difference in interaction between PHE and the membrane
and TRP could arise from many different factors either from
differences in monomer adsorption or possibly differences in
self-assembled structures, which could potentially adsorb
directly to the membrane. In a study of amino acid monomer
adsorption, phenylalanine interacted with a copper substrate
through its termini whereas tryptophan lay flat with the indole
moiety parallel to the substrate.16,17 While tryptophan has been
observed to aggregate in solution, it does not produce the
ordered chiral structure of phenylalanine at the temperatures
considered in their work.18
Understanding the nature of these self-assembled structures
can inform how they may interact with proteins, membranes
or other aggregates (whether ordered or disordered). If these
assemblies contain relatively fixed domains, they may share
some properties with molecular crystals even though they are in
solution.19 In molecular crystals, once a trace of a thermo-
dynamically preferred polymorph exists in a sample, it is often
difficult to produce other polymorphs even if they had been
previously observed. In the same way that multiple polymorphs
exist in crystal structures, multiple folding arrangements are
observed in certain proteins and one arrangement can provide
a seed for the selection of that polymorph or folding pathway.
In this way, the prion associated with Creutzfeld–Jakob syn-
drome can seed the for the formation of that polymorph which
progresses the course of the amyloid disease.19 This was
observed with amyloid-like phenylalanine fibrils recently where
the presence of pre-formed phenylalanine fibrils directed
other amino acids and even large proteins to assemble into
toxic fibrils.20 The ability of toxic assemblies to seed for and
promote other toxic assemblies reinforces the need to better
resolve the assembly processes and to inform rational thera-
peutic approaches.
From molecular dynamics simulations the differences and
similarities of nanostructures formed by single aromatic amino
acids can be observed with molecular level resolution. These
observations can be used to explain physical phenomena
observed in various experimental studies from multiple fields.
Mixtures of these molecules can then be simulated to predict
different microstructures expected to exhibit different behavior
compared to their corresponding pure systems.
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Methods
We have simulated the assembly of tyrosine (TYR) and trypto-
phan (TRP) following our earlier study on phenylalanine (PHE).
We also investigated mixtures of these amino acids in order to
explore potential relevance to disease mitigation. Our simula-
tions were performed using the GROMACS software,21 OPLS-AA
force field, and TIP3P explicit water molecules. The OPLS-AA
force field was selected as it provides good hydration properties
of amino acids,22,23 and so presents greater agreement to
experimental solubility where other force fields may under-
estimate.24 It was also used in studies on the effect of pH on
the assembly of small phenylalanine containing peptides.25
We account for the periodicity by using the Particle Mesh Ewald
(PME) algorithm for calculating electrostatic interactions.26,27
Consistent with our previous paper, velocities were generated
from a Maxwell distribution and initial configurations were
energy minimized using steepest descent minimization with
a 0.01 ps step size. Brief NVT and NPT equilibration runs
preceded each 300 ns simulation. The Berendsen thermostat
kept temperature constant and the Parrinello–Rahman algo-
rithm held the pressure at 1 bar.28 The integration step for all
simulations was 2 fs. Our simulations begin with 27 monomers
positioned randomly in a 5 nm cubic box. The 27 monomers
were selected to be consistent with a previous study4 and
results in a concentration of 360 mM. Because we chose to
simulate the individual amino acids in a neutral pH scenario,
we represented each molecule as an uncapped zwitterion.
Other studies have been performed in which termini are both
capped and uncapped and differences in molecular inter-
actions and assembly behaviour observed.29,30 The practice of
capping helps prevent the unwanted interactions of charged
termini when trying to simulate the behaviour of particular
amino acids within a larger protein.31 A relevant example would
be diphenylalanine as the recognition motif of the beta-amyloid
polypeptide in Alzheimer’s disease.32 However, since a number
of diseases are connected to the accumulation of individual
amino acid molecules (e.g. phenylalanine in PKU),4 we were
interested in the assembly of individual amino acids molecules
at physiological pH and thus have represented the termini
of each isolated monomer as positive (–NH3+) and negative
(–COO ).
Whereas our previous study utilized a simulated annealing
approach, this study involves constant temperature runs at 4
different temperatures (275 K, 300 K, 325 K, 350 K). The pure
systems, and then the binary mixtures, were simulated and free
energy landscapes were produced for each set of conditions.
To produce the free energy landscapes, an in-house clustering
algorithm reminiscent of the Hoshen–Kopelman algorithm was
applied to each simulation after the first 100 ns. A cluster was
defined as having 4 monomers with separation distances less
than 6 Å if multiple clusters were within 6 Å they were merged
together and considered as one cluster. Final clusters were
evaluated for the total number of monomers and the height
of the cluster. The height of the cluster was the greatest
distance between a-carbons within the cluster. The number of
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monomers and the height of each cluster are used as the x and
y axes and the z axis is the free energy as determined by
F= kT log(P(x,y))
Using this equation, free energy landscapes were produced for
the pure systems and each mixed system.
Results
Phenylalanine
We have previously reported the formation of a four-fold
structure via simulated annealing in which the zwitterionic
termini are internal to the tubular structure and aromatic
rings are in contact with the water molecules. In our current
simulations we observe this four-fold structure form at high
temperature. This four-fold structure and another ‘‘zig-zag’’
structure we observe throughout the study are shown in
Fig. 1. At 350 K, the four-fold symmetry is evident in one tube
and a second cluster interacts through aromatic rings to the
side of the predominant tube. The 325 K simulation resulted in
a much more rapid formation (within 60 ns) of the four-fold
symmetry compared to the higher temperature. At 300 K, a
similar rate of formation was observed but with less order
(determined by visual inspection). At 100 ns, the organization
of monomers developed into the zig-zag structure observed
previously that in many ways resembles phenylalanine’s crystal
structure. This persisted until the end of the simulation and
results in the free energy landscape typical of these systems.
As shown in Fig. 2, the minimum exists as a rectangular block
to the far right indicates a structure with large cluster numbers.
The height of the zig-zag structure has a fairly variable range
(B40 Å) compared to the tubular structures, which tend to have
a narrow range (B10–15 Å) of heights associated with minimal
energy. The structure that forms at 275 K is a short four-fold
structure where instead of having elongation in the direction of
the tubular axis, additional monomers were added to the
structure laterally as described by Do et al.33 In this particular
simulation a point of divergence between the four-fold struc-
ture and the zig-zag structure was observed at 180 ns in which
either structure could reasonably have formed (Fig. S1, ESI†).
There were four monomers interacting through termini like in
the four-fold structures but they were slightly offset to one
another similar to the zigzag structure. In this case monomers
added above/below the four-fold nucleus and formed a four-
fold structure with several layers. The same arrangement could
have very easily progressed toward the zig-zag structure had
nearby monomers approached from different directions/
orientations. In our previous work 4 out of 5 simulated annealing
runs resulted in four-fold tubes with the fifth forming the
‘‘zig-zag’’ structure giving fairly good reproducibility and relatively
quick structure formation in 300 ns. In Fig. 3, the hydrogen bond
formation is shown as a function of time for the constant

Fig. 1 Simplified examples of structures observed throughout the study. (a) Control showing no aggregation, (b) disordered aggregation, (c) some order
present (d) ordered structure in the commonly observed ‘‘zig-zag’’ form with the top view (left) and an angled view (right), and (e) ordered structure in the
commonly observed four-fold structure showing the side view (left) of the tube, and a top view (right).

Fig. 2 Free energy landscapes for pure phenylalanine (PHE) simulations at (a) 275 K, (b) 300 K, (c) 325 K, and (d) 350 K. Final structures are shown below
the landscape with phenylalanine displayed in red.

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Fig. 3 Number of hydrogen bonds between individual phenylalanine monomers as a function of time and temperature (left) and solvent accessible
surface areas of the monomer backbones as a function of time and temperature (right).
temperature phenylalanine self-assembly. At 300 ns it seems
that the assembly has not fully reached equilibrium, which
aligns with visual inspection. Therefore these systems were
extended an additional 100 ns. Also shown in Fig. 3 is the
decrease in solvent (water) accessible backbone area as a
function of time and temperature. As the assemblies form,
the charged termini are drawn close to each other, and away
from the solvent. At 400 ns we see these values beginning to
level indicating more complete formation. This demonstrates
the simulated annealing is a more efficient approach to achieving
the fully formed systems but that given a longer timescale,
constant temperature can also provide substantial insight.
Tyrosine
At 350 K, tyrosine exhibited very quick aggregation as an
ordered structure. A single layer of four grew to several resulting
in a short three-layer, four-fold tube interacting with another
short tube through the aromatic rings. In other systems this
orientation may well have persisted however, with tyrosine this
structure was short-lived and instead rearranged to a single
elongated tube with a symmetry of four. This can be attributed
to the hydroxyl group making the aromatic ring more polar and
therefore more willing to be exposed to water molecules than,
for example, phenylalanine under the same constant tempera-
ture conditions. The formation of this four-fold structure is also
in agreement with some previous simulation work performed
on pure tyrosine.9 At 325 K, aggregation happens fairly quickly
but there is a lag time until order develops. In this case the
order is in the form of the zig-zag structure, which takes on the
typical rectangular shaped-minima shown in Fig. 4. At both
high temperatures these higher ordered structures are observed
however additional replicates (Fig. S3 and S4, ESI†) show that
which structure forms is sensitive to an element of probability
similar to what was described for phenylalanine assembly.
At the two lower temperatures less order is observed in general.
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At 300 K, four-fold symmetry is observed but only transiently
and aggregation occurs but not with any significant order
persisting through the simulation. At the end of the simulation
two ladder-like systems parallel to one another are present and
interact laterally through aromatic rings. At 275 K, there is
aggregation but, again, a lag time before order develops. In this
case, the system has a fairly long tube formed but only one four-
fold layer exists and is perturbed by the C–N termini direction-
ality. When neighboring strands possess opposite termini
directionality, distinct layers of four are present as each termini
is in closest contact with two of the oppositely charged termini.
When this is not the case and two neighboring strands possess
the same directionality, the proximity of like functional groups
is unfavored and so forces a shift of one strand relative to the
next. While this is quite intuitive energetically, it has the
consequence of producing ‘‘layers’’ that have the four-fold
symmetry from the top but that do not present as distinct
layers from the side view as shown in Fig. S2 (ESI†). The high
temperatures favoring ordered structures is a very interesting
observation especially for tyrosine which self-assembles as
a monolayer on a silver surface only above a temperature
threshold of 320 K.34 At 350 K, we observe tyrosine adopt the
four-fold structure that we first presented for phenylalanine.
It should be noted that the concentrations are held constant
throughout this study for direct comparison but that tyrosine
has much lower water solubility than phenylalanine or
tryptophan. Fig. 5 shows the assembly process progress as a
function of time and temperature. The termini become buried
in the aggregate and therefore are no longer as accessible to
water and the same time as hydrogen bonds are formed
between the NH3+ and COO functionalities. It is distinctly
clear in this result that this process occurs fastest for the
highest temperature (350 K, blue lines), followed by 325 K
(green lines) and that it occurs more slowly in the case of the
lower temperatures (red and black).
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Fig. 4 Free energy landscapes for pure tyrosine (TYR) simulations at (a) 275 K, (b) 300 K, (c) 325 K, and (d) 350 K. Final structures are shown below the
landscape with tyrosine displayed in green.
Fig. 5 Number of hydrogen bonds between individual tyrosine monomers as a function of time and temperature (left) and solvent accessible surface
areas of the monomer backbones as a function of time and temperature (right).
Tryptophan
At high temperature (350 K), tryptophan aggregates very quickly
into a four-fold structure, which gains monomers throughout
the simulation and ends with an impressively neat four-fold
tubular structure. At 325 K, the aggregation process is quick but
there is a lag time before order develops in the system com-
pared to the higher temperature simulation. At the end of the
simulation, two tubes with symmetry of four exist interacting
through the aromatic rings. This is in contrast to the higher
temperature result of tyrosine where the tubes interaction through
the rings was a transient state replaced by an elongated tube with
more ring-water contact. With tryptophan, the large hydrophobic
rings may prefer to be buried from the water and so form these
laterally interacting clusters. At 300 K, aggregation occurs but with
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a distinct lack of order throughout the simulation. This is evident
in Fig. 6 where lots of monomers are visibly clustered but with no
clear alignment of termini or rings. At 275 K, the aggregation
process is sluggish compared to higher temperatures (also see
Fig. 7) and there is a similar lack of order throughout the
simulation as observed at 300 K. It is apparent that the high
temperature promotes both the aggregation and the degree of
organization of the tryptophan self-assembly. The fact that trypto-
phan does not form structures with a high degree of order near
room temperature is consistent with circular dichroism studies.18
Mixtures
Having observed the assemblies of each pure amino acid
system at various temperatures, we sought to expand our study
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Fig. 6 Free energy landscapes for pure tryptophan (TRP) simulations at (a) 275 K, (b) 300 K, (c) 325 K, and (d) 350 K. Final structures are shown below the
landscape with tryptophan displayed in blue.
Fig. 7 Number of hydrogen bonds between individual tryptophan monomers as a function of time and temperature (left) and solvent accessible surface
areas of the monomer backbones as a function of time and temperature (right).
to mixtures of amino acids (Fig. 8–10). Mixed systems have the
potential of completely changing the nature of the fibrils. When
D-phenylalanine was added to L-phenylalanine the course of
self-assembly was completely altered with potential implications
in disrupting the course of a disease caused by phenylalanine
assembly.35 Doxycycline was observed to modify the growth of
phenylalanine fibers.36 Most recently, several polyphenol struc-
tures were observed to disrupt phenylalanine fibril formation in
a dose-dependent manner.37 These mixed system furthered our
interest for potential biological consequences.
At 350 K, the system containing all tryptophan contains four
fully formed layers with the symmetry of 4. The replacement of
5 monomers with phenylalanine does not have an effect on the
structure formed. With 10 phenylalanine molecules there are
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5 fully formed layers and the length of the fibril is sufficiently
long to begin to observe slight tortuosity. When 14 molecules of
phenylalanine were simulated with 13 molecules of tryptophan
the symmetry of four was still observed but a second isolated
cluster of 5 was present with a different organization. Seventeen
phenylalanine and ten tryptophan molecules formed a struc-
ture more reminiscent of the zigzag structure than the four fold
structure. With 22 molecules of phenylalanine, the zig-zag
structure was observed with tryptophan impurities. With pure
phenylalanine the symmetry of four was formed and while
elements of four layers of four were present, the end layers
were incomplete. Additionally, seven monomers were asso-
ciated as a second imperfect fibril with fibril axes rotated from
alignment.
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Fig. 8 Free energy landscapes for mixtures of tryptophan (TRP) and phenylalanine (PHE) at 275 K, 300 K, 325 K, and 350 K. Final structures are shown
beside the landscapes with tryptophan displayed in blue and phenylalanine in red.

Fig. 9 Free energy landscapes for mixtures of tyrosine (TYR) and phenylalanine (PHE) at 275 K, 300 K, 325 K, and 350 K. Final structures are shown below
the landscape with tyrosine displayed in green and phenylalanine in red.

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Fig. 10 Free energy landscapes for mixtures of tyrosine (TYR) and tryptophan (TRP) at 275 K, 300 K, 325 K, and 350 K. Final structures are shown below
the landscape with tyrosine displayed in green and tryptophan in blue.
For the tryptophan-phenylalanine mixtures at 325 K, the
four-fold structures that form at high mole fractions phenyl-
alanine suffer from parallel strand alignment (example shown
in Fig. S2, ESI†). When neighboring strands possess opposite
termini directionality, distinct layers of four are present as each
termini is in closest contact with two of the oppositely charged
termini. When this is not the case and two neighboring strands
possess the same directionality, the proximity of like functional
groups is unfavored and so forces a shift of one strand relative
to the next. While this is quite intuitive energetically, it has the
consequence of producing ‘‘layers’’ that have the four-fold
symmetry but are not clear-cut entities. This results in less
distinct layers but also seems to increase the flexibility of strands.
At 275 K, the pure phenylalanine system has two parallel
four-fold structures interacting laterally as described by Do
et al.7 The addition of only 5 monomers of tryptophan results
in a transition from the four-fold system to a disordered
aggregate. In general, it seems that at low temperatures, more
order exists in pure phenylalanine than for tryptophan, con-
sistent with experiment.18 Under these conditions, addition of
tryptophan results in a considerably less ordered system.
At 350 K, we observe the four-fold structure with an indication
of lateral growth preferred over growth in the axis’ direction.
With the addition of small amounts of tryptophan the system
converts to the zig-zag. With further addition of tryptophan,
even better formed four-fold structures are observed where the
direction of growth occurs along the axis. As some degree of
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sensitivity is observed in the case of pure systems it is best to
consider these mixed systems as ‘‘ordered’’ or disordered
aggregates.
At high temperature both tyrosine and phenylalanine pro-
duced structures with four-fold symmetry. Unsurprisingly,
every combination of these two molecules at 350 K contains
this arrangement. In both pure systems, decreasing in tem-
perature resulted in less well-ordered assemblies. Similarly, the
mixed systems at lower temperatures showed less order than
their higher temperature counter-parts. At 350 K four-fold tubes
are observed at each mole fraction. At 325 K the zig-zag
structure is seen for pure tyrosine and fairly equimolar mixtures
of the two amino acids. All other mole fractions contain four-fold
tubes. The zig-zag structure becomes more prominent at 300 K.
At the lowest temperature, 275 K, disordered aggregates are
observed with the exception of the equimolar system for which
the zig-zag structure is present.
Both tyrosine and tryptophan form four-fold systems in their
pure assemblies at 350 K. Higher tryptophan content and lower
temperatures favor the formation of the disordered aggregates
seen in the pure tryptophan systems. The larger hydrophobic
region of tryptophan molecules would be expected to increase
the self-assembly behavior due to the entropically driven
hydrophobic effect. In each simulation we observe tryptophan
molecules forming assemblies as expected but the degree of
order seems very much dependent on temperature. It has been
suggested that as peptide assemblies form and take on more of
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a solid-state character they can be reasonably compared to mole-
cular crystals for which enthalpic factors play a large role.19
Discussion
One of the most striking observations in this study was the
fact that higher temperature in the case of each amino acid
promoted more ordered structures. We explain this on the
basis of diminished hydrogen bonding between termini and
water molecules. There is great agreement in the literature to
suggest that the average number of hydrogen bonds that water
molecules are capable of forming, decreases with increasing
temperature.38–40 In a system where hydrogen bonding between
termini and water molecules is in competition with direct
electrostatic termini interaction, the increasing temperature
suppresses the role of hydrogen bonding between water mole-
cules and termini and allows the interactions between termini
of neighboring amino acids to become more dominant. As a
consequence, we observe the termini interactions form more
readily at high temperatures. To capture the interaction between
water molecules and different portions of the amino acids, the
solvent accessible surface area was calculated and shown for each
pure system as a function of temperature (Fig. 3, 5, 7, and 12). In
particular, the interaction between water and the termini (SASA of
the Backbone) decreases as a function of time and this occurs
much more dramatically at higher temperatures, in line with the
previously stated hypothesis. At the same time, as the interaction
between the amino acid backbone and water is suppressed,
we show the hydrogen bond formation between amino acid
molecules (Fig. 3, 5, and 7). This, again, happens more dramati-
cally at higher temperatures as the number of interactions water
molecules can sustain diminishes.
Another observation was that the phenylalanine system seemed
to have not reached full self-assembly although indications of
four-fold vs. zig-zag were apparent at 300 ns, these runs were
extended to 400 ns. Our previous work5 utilized a simulated
annealing approach and at the 300 ns time-point, fully formed
nanostructures had already been produced. In particular, 4 out of
5 replicates showed the four-fold symmetry and the remaining
trajectory formed the zig-zag structure (example in Fig. 1). This
potential to form different structures under the same conditions
was described previously and is again observed in our current work
(Fig. S1, ESI†). To probe this further under constant temperature
conditions, we selected conditions that produced a zig-zag struc-
ture and a four-fold tube and ran multiple replicates of the same
conditions (Fig. S3–S5, ESI†). We found that we can quite repro-
ducibly determine when an ordered structure (zig-zag or tube) will
form but cannot yet reproducibly predict which structure will
form. Consistently producing one or the other nanostructure is
not achieved under our current procedure but future testing may
determine an additional parameter (concentration, salt, pH, other
compositional changes) that can provide this desired tuning of the
nanostructure.
In addition to being able to confidently determine when an
ordered structure forms versus a disordered aggregate (e.g. TRP
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Fig. 11 Final 300 ns snapshots of 27 alanine molecule simulations at each
temperature considered in this study; 275 K, 300 K, 325 K, and 350 K.
at low temperature in agreement with experiment18), we have
also observed a non-aromatic amino acid fail to aggregate at all.
In Fig. 11 we show the results of alanine controls performed at
the different temperatures used in our study. Alanine lacks the
large aromatic functionalities of phenylalanine, tyrosine, and
tryptophan and is commonly used as a negative control in
related experimental work.4,10,36 Without these large hydro-
phobic functionalities, we observe that alanine does not form
aggregates but rather the molecules remain dispersed through-
out the simulations (Fig. 11). This emphasizes the importance
of the hydrophobic portion of the molecules and confirms
that while electrostatics (Fig. S5, ESI†) and hydrogen bond
formation play a role in the assembly process, they are insuffi-
cient on their own to produce the ordered structures we observe
with phenylalanine, tyrosine, and tryptophan. This, and the
proximity of aromatic rings in our final nanostructures suggest
that hydrophobic interactions including p–p stacking play an
important role. In line with the hydrophobic effect,41 we show
in Fig. 12 that the solvent accessible surface area of the
side chains of the amino acids decrease as a function of time.
As the rings come together along the length of the assembly,
the SASA indicates that water molecules are excluded from
these hydrophobic regions and are then free to diffuse throughout
the bulk.
Membrane interaction of phenylalanine and related
compounds have been described in the literature to gain funda-
mental understanding,42–44 to understand how phenylalanine
fibrils impact hemolysis in PKU patients,20 and to understand
membrane stability of freeze resistant plants.15 Popova et al.
found that both tryptophan and phenylalanine cause membrane
leakage and result in toxicity to plant cells.15 While the authors
had no reason to consider aggregate formation playing a role in
the toxicity, their work is performed in the same concentration
range as the work produced by Abramovich et al. and it is entirely
possible that membrane disruption they observe was due to the
interaction of amino acid fibrils and not monomers. The concept
of fibrils impacting membrane function is not unfounded. Several
studies have described fibrils disturbing membrane function and,
in particular, this has been seen with amyloid fibrils.20,45–47
A separate study prepared phenylalanine using a freeze-drying
technique within the same temperature range. They were
interested in controlling the nature of the phenylalanine fibrils
and observed short fibril formation at 10 mM and straight,
well-aligned fibrils at 100 mM.9 This study, at comparably low
temperature, further suggests that the membrane damage in
the Popova et al. study could have been caused by phenyl-
alanine fibrils.
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Fig. 12 Solvent accessible surface area of the hydrophobic regions for phenylalanine, tyrosine, and tryptophan at different temperatures: 275 K (black),
300 K (red), 325 K (green), and 350 K (blue).
The preference of tyrosine and tryptophan to form the four-
fold assembly is increased by high temperature in our simula-
tions, which is also supported by some experimental evidence.
Tryptophan assemblies at room temperature do not form
ordered systems or this would have been evidenced by circular
dichroism studies.18 Tyrosine was observed to form fibrils on a
silver surface above a threshold of 320 K.34 In separate work,
tyrosine fibrils were formed at high concentrations. To reach
the high concentrations necessary to form the amyloidogenic
fibrils, a heating step at approximately 360 K was required.10,48
Due to the potential impact of mixed systems on altering
disease pathways, it was important to investigate the mixtures
of these systems. The mixtures of aromatic amino acids
produced a variety of structures with the four-fold tube and
the zig-zag structure being the two ordered systems common to
the aromatic amino acids. In addition to the binary systems
described above, we performed simulations of equimolar
mixture of phenylalanine, tyrosine and tryptophan and also
saw four-fold fibril formation at high temperature. Bibin Anand
et al. demonstrated that taking 15% w/w phenylalanine fibrils
(formed at 6 mM) resulted in intense aggregation behavior with
a mixture of 0.1 mM amino acids including tyrosine and
tryptophan. This seeding behavior was also shown for larger
proteins and has significant implications for the understanding
of PKU.
Unless the four-fold tube is considered from a purely
physical packing standpoint, it is indeed surprising. In related
self-assembly literature, Israelachvili describes a packing para-
meter that predicts how amphiphilic compounds tend to
arrange upon self assembly.49 This parameter is ratio of the
volume of the hydrophobic tail compared to the area of the
hydrophilic head times the length of the fully extended tail.
In many systems, this packing parameter is less than one, and
a variety of assemblies can be predicted including spherical
30534 | Phys. Chem. Chem. Phys., 2018, 20, 30525--30536
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PCCP
micelles and cylinders. In each case, when this parameter is
less than one, the hydrophilic heads are on the outside of the
assembly with hydrophobic tails on the inside. When this ratio
is greater than one (i.e. in the case of sufficiently bulky hydro-
phobic tails), inverted structures like those in our simulation
results are predicted. Without the packing consideration the
result is indeed counterintuitive but also may explain how this
particular nanostructure may produce the toxic effects
observed in phenylketonuria and other aromatic amino acid
related diseases. While these structures were a bit unexpected,
this actually makes them well-suited for membrane insertion
and resulting leakage.6 In this work we have demonstrated the
formation of this potentially toxic nanostructure in tyrosine
and tryptophan as well. We have also described a second
re-occurring nanostructure, the zig-zag structure. This nano-
structure may present a non-toxic form of the assembly or it
may have even greater biological implications. If switching
between one nanostructure to another impacts the eventual
disease state, this will be worth additional study and compar-
ison to experimental work. Many studies have shown the
differences in microstructures as a function of solvent
content9,50,51 and ion content9 and these present valuable
considerations for future work. It is possible that one of these
factors and/or the addition of therapeutics will result in
consistent production of one structure over another in a way
that we have yet to achieve.
Conclusions
At high temperatures phenylalanine formed the four-fold
tube previously observed in simulated annealing. At lower
temperatures lateral growth was preferred which resulted in
less exposure of aromatic rings to the explicit water molecules.
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For tyrosine, the high temperature simulation also resulted in
the four-fold structure and with a greater propensity to grow in
the direction of the axis leaving more aromatic rings (in this
case with the hydroxyl group) exposed to water. At low tem-
perature these molecules did not self assemble as readily and
did not form very ordered structures. Tryptophan showed a
similar result with high temperatures promoting extremely well
organized fibrils and low temperature essentially disordered
aggregation was observed. Intermediate temperatures and
compositions resulted in a variety of structures including a
common zig-zag structure and four-fold tubes that grew either
vertically or laterally.
Conflicts of interest
Helen W. Hernandez is a Managing Member of KAL Research
Initiatives, LLC.
Acknowledgements
This work was supported by the scientific research project
2016BF0018 of Turkish-German University.
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