Aliphatic peptides show similar self-assembly to
amyloid core sequences, challenging the importance
of aromatic interactions in amyloidosis
Anupama Lakshmanana,1, Daniel W. Cheongb,1, Angelo Accardoc, Enzo Di Fabrizioc, Christian Riekeld,
and Charlotte A. E. Hausera,2
aInstitute of Bioengineering and Nanotechnology, Singapore 138669; bInstitute of High Performance Computing, Singapore 138632; cIstituto Italiano
di Tecnologia, 16163 Genoa, Italy; and dEuropean Synchrotron Radiation Facility, 38043 Grenoble Cedex 09, France
Edited* by Alexander Rich, Massachusetts Institute of Technology, Cambridge, MA, and approved November 21, 2012 (received for review October 12, 2012)
The self-assembly of abnormally folded proteins into amyloid fibrils
is a hallmark of many debilitating diseases, from Alzheimer’s and
Parkinson diseases to prion-related disorders and diabetes type II.
However, the fundamental mechanism of amyloid aggregation
remains poorly understood. Core sequences of four to seven amino
acids within natural amyloid proteins that form toxic fibrils have
been used to study amyloidogenesis. We recently reported a class
of systematically designed ultrasmall peptides that self-assemble in
water into cross-β–type fibers. Here we compare the self-assembly
of these peptides with natural core sequences. These include core
segments from Alzheimer’s amyloid-β, human amylin, and calcitonin.
We analyzed the self-assembly process using circular dichroism,
electron microscopy, X-ray diffraction, rheology, and molecular
dynamics simulations. We found that the designed aliphatic peptides
exhibited a similar self-assembly mechanism to several natural
sequences, with formation of α-helical intermediates being a
common feature. Interestingly, the self-assembly of a second core sequence
from amyloid-β, containing the diphenylalanine motif, was
distinctly different from all other examined sequences. The diphenylalanine-
containing sequence formed β-sheet aggregates without
going through the α-helical intermediate step, giving a unique fiberdiffraction
pattern and simulation structure. Based on these results,
we propose a simplified aliphatic model system to study amyloidosis.
Our results provide vital insight into the nature of early intermediates
formed and suggest that aromatic interactions are not as
important in amyloid formation as previously postulated. This information
is necessary for developing therapeutic drugs that inhibit
and control amyloid formation.
Amyloid fibril formation is implicated in a wide range of
chronic degenerative diseases such as Alzheimer’s, Parkinson,
and prion-related diseases, as well as others, such as diabetes
type II (1). Although amyloidogenic proteins from unrelated
diseases do not share any sequence homology, the structural
properties of the amyloid fibrils are similar (2). Non–diseaseassociated
proteins can also form amyloids in vitro, suggesting
that the process is ubiquitous (3). Despite the clinical significance
of amyloidogenesis and its widespread occurrence, the
exact mechanism of amyloid formation remains unclear. Several
studies have indicated that amyloids, commonly characterized by
a highly ordered cross-β structure (4), are associated with proteinspecific
amyloidogenic core sequences of a few amino acids (2, 5,
6). These core sequences, usually four to seven amino acids long,
are able to self-assemble into fibrils physically, morphologically,
and tinctorally similar to those observed with the entire protein
(2, 6–9). Thus, they have the potential to serve as model systems,
simplifying the study of amyloidosis. Furthermore, there is
growing evidence that early oligomers during the self-assembly
process are more pathogenic and toxic compared with mature
fibrils (10, 11). Clearly, there is an urgent need to identify the
structures and properties of the early oligomers to arrest molecular
recognition and self-assembly in the earlier stages.
Recently, we reported a class of rationally designed ultrasmall
peptides that self-assemble in water into amyloid-β−type fibers
(12). Based on this, we proposed a mechanism for amyloid formation
involving a conformational transition of the structurally
unorganized monomers into metastable α-helical intermediates
that terminate in cross-β structures (12). The current study
compares the self-assembly process of these peptides with naturally
occurring amyloidogenic core sequences from three amyloid-
associated diseases, namely Alzheimer’s, diabetes type II,
and thyroid medullary carcinoma. We aim to extend the self-assembly
mechanism observed for rationally designed peptides to
natural amyloidogenic sequences. We studied the self-assembly of
designed and natural peptide sequences using circular dichroism,
electron microscopy, X-ray fiber diffraction, rheology, and molecular
dynamics simulations.
An increased occurrence of aromatic residues in natural core
sequences has led to widespread conclusions about the crucial
role played by these residues in molecular recognition and selfassembly
(13–15). Comparing the self-assembly of our fully aliphatic
designed peptides with natural core sequences would also
help to determine the significance and effect of π–π interactions
on amyloid formation.
In addition, we wanted to identify and characterize key early
intermediates that drive self-assembly and can thus serve as
a common target across diseases for therapeutic targeting and
intervention.
Results
The investigated rationally designed peptides (LD6 and ID3),
natural amyloidogenic core sequences (NL6, DF5, GA6, and
KE7), and RADA are listed in Table 1. Their origins and specific
secondary structural transformations during self-assembly are
also given. LD6 (LIVAGD) and ID3 (IVD) are two extensively
characterized ultrasmall peptides (12, 16), namely the bestperforming
6-mer with respect to propensity of gelation and gel
strength, and a 3-mer. The natural core sequences include defined
“hotspots” within amyloid proteins of high amyloidogenic potential,
without and with one or two aromatic residues at different
positions in the sequence (2, 5–8, 14, 17). RADA is an ionic selfcomplementary
16-mer, known for forming stacks of flat β-sheets
(18). All peptides were acetylated at the N terminus, with RADA
additionally amidated at the C terminus.
Amyloidogenic Core Sequences Self-Assemble into Similar Structures
as LD6 and ID3. NL6, DF5, GA6, and KE7 as well as LD6 and ID3
self-assembled into hydrogels (Fig. S1 A–F). GA6 (GGVVIA) is
Author contributions: C.A.E.H. designed research; A.L., D.W.C., A.A., C.R., and C.A.E.H.
performed research; E.D.F. contributed new reagents/analytic tools; A.L., D.W.C., C.R., and
C.A.E.H. analyzed data; and A.L., D.W.C., C.R., and C.A.E.H. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1A.L. and D.W.C. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: chauser@ibn.a-star.edu.sg.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1217742110/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1217742110 PNAS Early Edition | 1 of 6
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an aliphatic core sequence from the transmembrane domain of
amyloid-β (Aβ) with a high propensity for amyloid aggregation
(17). Another core sequence is KE7 (KLVFFAE), containing the
sequence LVFFA (19) with its diphenylalanine (FF) motif,
considered essential for aggregation of Aβ (15, 20). There is a
minimum critical concentration for each peptide below which no
gelation is observed. Above the critical value, the speed of hydrogel
formation followed the order LD6 ≅ DF5 > NL6 > GA6 >
KE7 > ID3, with the order LD6 > ID3 as previously observed
(12, 16). Formation of a solid gel was determined by visual inspection
(turning the vial upside down). At 12 mM, LD6 formed
the most shape-compact and homogeneous gel, whereas NL6,
GA6, and DF5 showed slight phase separation of the fibrous
aggregates from the aqueous phase (Fig. S1 A–F). KE7 formed
hydrogels and aggregates with different properties that will be
described below.
The morphology of fiber networks constituting the hydrogels
was evaluated by field emission scanning electron microscopy
(FESEM) (Fig. 1 A, B, and D–F). Interestingly, the natural
sequences NL6, DF5, and GA6 formed fiber networks with similar
morphology to the aliphatic peptides, with helical twists and
turns observed for individual fibers as well as formation of fiber
bundles (Fig. 1 A, B, and D–F). The SEM image of the NL6
residue attached to a glass capillary showed a corrugated morphology
(Fig. S2A), whereas the higher-resolution SEM image
revealed fibers with periodic helical turns (Fig. 1C).
X-Ray Fiber Diffraction of NL6. Raster diffraction of the NL6 residue
dried on a superhydrophobic substrate revealed fibrous
aggregates (Fig. 2A), which were also visible in the electron
microscope images (Fig. 1 C and D and Fig. S2A). The convective
flow-induced nanofibrillar alignment in the drying residue
resulted in diffraction patterns with fibrous texture (21) revealing
a cross-β pattern (Fig. 2B) (22–24). The azimuthally averaged
intensity profiles of the meridian and equator are shown in Fig. 2
C and D. We identified on the meridian a 0.46-nm peak as
second-order β-strand periodicity for a 0.92-nm period (a axis)
(Fig. 2C). The weak 100 reflection was assumed to be part of the
asymmetric 1.02-nm peak, which also contains the tails of an
equatorial peak. A broad 0.40-nm peak was attributed to shortrange
ordered β-sheet material. The equatorial 0.97-nm reflection
(Fig. 2D) corresponded to the correlation found along
the chain direction (b axis) in amyloid structures. The >0.97-nm
spacings observed on the equator were attributed to macrolattice
ordering of β-crystallites along the chain-axis (b) direction, for
example due to a helical twist (25). We attributed the reflections
to 0k0 orders with 2.07 nm (k = 1), 0.97 nm (k = 2), 0.66 nm (k =
3), 0.34 nm (k = 6) for a 1.96(2) nm b axis (average of orders 2/3)
(Fig. 2 C and D). The b-axis dimension corresponds nearly to
a fully extended 6-mer peptide chain of 0.35 × 6 = 2.1 nm. The
equatorial 0.66-nm peak was attributed to the c-axis 001 reflection
(intersheet direction) (Fig. 2C). This suggests an orthorhombic
unit cell with a = 0.92 nm, b = 1.96 nm (chain direction),
c = 0.66 nm, α = β = γ = 90°.
Rheology. Characterization of the different peptide hydrogels was
done by oscillatory rheology. ID3 was not included, due to its
smaller size compared with the other peptides. To ensure comparability
and standard sample preparation for all the peptides,
the ring-cast method was used (SI Materials and Methods). Surprisingly,
LD6 formed the stiffest, most shape-compact, and
homogeneous gel compared with all examined peptides (Fig. 3 A
and B and Fig. S1). This is reflected by the high elastic modulus
(G′ value) of over 104 Pa and the visual appearance of the gel,
which assumes the well-defined shape of the mold (Fig. 3A). In
addition, no separation of the fibrous aggregates from the aqueous
phase was observed. The G′ value for LD6 is higher than that
of RADA, an ionic self-complementary 16-mer peptide, at the
same molar concentration (18). In contrast to LD6, KE7 formed
a weak viscous gel with the lowest G′ value (Fig. 3A). In all cases,
the trend of G′ values supported our empirical observation on the
stiffness of the hydrogels. The linear viscoelastic range (LVR) was
similar for LD6, NL6, DF5, and GA6 (Fig. 3B). KE7, however,
showed a higher LVR, with a yield point similar to that of RADA
and different from the other peptides (Fig. 3B). The G′′ values of
the peptides also showed the same trend as the G′ values, with
LD6 being the highest and KE7 the lowest (Fig. S1G). The Tan
(δ) value for all peptide hydrogels was lower than 1, showing
dominance of the G′ (solid-like) component over the G′′ (fluidlike)
component.
Table 1. Sequences of investigated peptides
Peptide sequence Origin (associated amyloid disease) (ref.)
α-Helical
intermediates
Helical
fibers/β-turns
LIVAGD (LD6) Designed synthetic peptide (12) Yes Yes
IVD (ID3) Designed synthetic peptide (12) Yes Yes
NFGAIL (NL6) Human amylin (diabetes type II) (8) Yes Yes
KLVFFAE (KE7) Human amyloid-β (1–42) (Alzheimer’s) (7) No No
GGVVIA (GA6) Human amyloid-β (1–42) (Alzheimer’s) (5, 17) Yes Yes
DFNKF (DF5) Human calcitonin (thyroid medullary carcinoma) (2) Yes Yes
RADARADA RADARADA (RADA) Designed synthetic peptide (18) No No
This table includes rationally designed ultrasmall peptides and core sequences of five to seven amino acids from naturally occurring amyloid proteins.
All peptides were acetylated at the N terminus.
Fig. 1. Morphological characterization of the peptide hydrogels using FESEM.
(A) ID3 (12 mM) ata magnification of 30,000×. (B) LD6 (12 mM) at 50,000×. (C)
High-resolution SEM image of NL6 attached to a glass capillary. (D) NL6 (12mM)
at 25,000×. (E) GA6 (12 mM) at 50,000×. (F) DF5 (12 mM) at 50,000×.
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1217742110 Lakshmanan et al.
Molecular Dynamics Simulations. Self-assembly of the peptides
NL6, ID3, and LD6 was individually investigated from simulations
of 64 molecules in a cubic box of water. Cube edge dimension
was 6.8 nm for ID3, 8 nm for LD6, and 8.2 nm for NL6.
(Movies S1, S2, and S3). Because the peptides are very short, it
was possible to observe self-assembly from our all-atom simulations.
Furthermore, the final concentrations of the peptide
solutions (Table S1) are much higher than that encountered
under experimental conditions, as we assumed that a higher
concentration would speed up the dynamics of the aggregation.
It is important to note that amyloidosis occurs over a much longer
period of many years within the human body and, therefore, a
higher starting concentration would be required to form the
aggregates within the simulation timescale. The simulations were
performed for 50 ns, except for NL6. NL6 revealed a highly disordered
aggregate at the end of 50 ns, and thus the simulation
was extended for an additional 50 ns to a total of 100 ns. Interestingly,
all three peptides self-assembled from monomers into
a partially organized but stable fiber (Fig. 4 A–C). Accounting for
periodic boundary conditions, the peptides effectively formed an
infinitely long fiber along a single axis (Fig. 4 A–C).
Fig. S5 A–C shows the density maps along the cross-section of
the fiber, giving an estimate of the fiber dimensions. The trimer
ID3 formed a relatively compact fiber that is more cylindrical in
shape, with a fiber diameter of around 2.7 nm. LD6 and NL6 also
formed clear fibers, albeit less ordered and compact. These peptides
exhibited a more ellipsoidal cross-section, with diameters
consistent with the relative length of the peptides. We performed
three independent simulations for each peptide to ensure that
different simulation trajectories result in similar aggregate morphology.
All simulations resulted in partially organized but stable
fibers. The cluster size distribution of the peptides over the course
of the 50-ns simulations was calculated to gain insights into the
dynamics of the self-assembly (Fig. S3 A–C). All three peptides
formed a single, stable aggregate fairly rapidly, with LD6 showing
the fastest dynamics where a single stable fiber was formed at 12–
13 ns. NL6 formed a single cluster at around 20 ns, whereas the
ID3 peptides formed a single cluster at around 28 ns. This is as
expected, because NL6 and LD6 have longer hydrophobic tails,
which increase the kinetics of aggregation. The cluster size distribution
also shows relatively few clusters of sizes between 10
and 64. This suggests that a minimum seed size of around 10
peptides is necessary for oligomeric cluster formation, after
which rapid growth to the single large cluster occurs. Further
studies are needed for confirmation.
Structural Transition via α-Helical Intermediates Points to a Common
Feature of Amyloidosis. Based on our published hypothesis of selfassembly,
the early initiation steps involve antiparallel pairing of
two peptide monomers and structural transition to an α-helical
dimer. The helical dimers then assemble into β-type fibers before
further condensation to cross-β aggregates (12). Surprisingly,
natural core sequences NL6, DF5, and GA6 showed similar random
coil to α-helix to β-turn transitions with increasing peptide
concentration (Fig. 5). The α-helical intermediates were characterized
by a negative π–π* transition near 222 nm, a negative split
π–π* transition near 208 nm, and a positive peak near 192 nm
corresponding to a π–π* transition (12). DF5 in particular formed
very stable α-helical intermediates, which appeared within
minutes at concentrations below 100 μM (Fig. 5C).
KE7 Shows a Different Fiber-Diffraction Pattern, Morphology, and
Self-Assembly Mechanism. Interestingly, KE7 (KLVFFAE) with
its FF motif not only formed aggregates and hydrogels with
distinctly different properties but also showed a unique X-ray–
diffraction pattern related to the convective flow alignment of
the evaporating droplet on the superhydrophobic substrate (Fig.
6 A–C). The gelation capacity and mechanical stiffness of the
formed hydrogel were notably lower than that of LD6 and the
Fig. 2. X-ray fiber diffraction of NL6. (A) Composite diffraction image of NL6
residue based on a raster-diffraction scan with 1-μm step increment. (B) A
single “pixel” at the position marked by an arrow in A corresponds to a fiberdiffraction
pattern. The meridional (m) and equatorial (e) directions are indicated.
(C) Meridional intensity profile fitted by three Gaussian profiles and
a 0-order polynomial. The d = 1.02 nm peak contains both meridional and
equatorial components. (D) Equatorial intensity profile fitted by five Gaussian
profiles and a 0-order polynomial. The experimental data in red were not used
for the fit (d values in nanometers). The fit of the 2.07 nm peak is less precise
due to the low-angle intensity tail, and the 0.34 nm peak due to several
overlapping peaks.
Fig. 3. Rheological characterization of peptide hydrogels at a concentration
of 12 mM. (A) Frequency sweep studies (storage modulus G′ as a function of
angular frequency ω) show that LD6 forms a shape-compact gel with the
highest mechanical stiffness and KE7 forms a weaker gel with lowest mechanical
stiffness. (Inset) LD6 (Left) and KE7 (Right) gels made by the ring-cast
method. (B) Amplitude sweep studies (storage modulus G′ as a function of
strain γ) show a similar LVR profile for peptide hydrogels with helical fiber
networks and a broader LVR region for KE7 and RADA. At least five independent
measurements (n = 5) were taken for each peptide.
Lakshmanan et al. PNAS Early Edition | 3 of 6
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other natural core sequences (Fig. 3A and Fig. S1). This could be
attributed to the formation of apparently short and flat nanotapes
rather than nanofibers (Fig. S2C). In addition, the high-resolution
SEM image showed no fibrillar morphology like NL6 (Fig. 1 C
and D) but rather a lamellar organization (Fig. 6F and Fig. S2B).
In contrast to other investigated core sequences, the circular
dichroism (CD) spectra of KE7 showed no α-helical intermediates
(Fig. 6E). With increasing concentration, a direct random
coil to β-sheet transition was observed, but no β-turn. The
β-sheet structure and molecular packing as inferred from the flat
nanotapes and lamellar organization could explicate the low
mechanical stiffness of KE7 hydrogels (Fig. 3A and Fig. S1G).
The lamellar layers slide over each other, unlike dense helical
fiber networks of other peptides that exert higher resistance to
applied shear. Furthermore, KE7’s LVR was very similar to that
of RADA and different from that of the other peptides (Fig. 3B
and Fig. S1H). RADA also forms a β-sheet structure and shows
a layer-like morphology by FESEM (Fig. S2D).
The composite diffraction image obtained by raster diffraction of
the KE7 residue showed mainly domains defined by weakly textured
∼0.47-nm β-sheet reflections (Fig. S4A).Based on themodel of slablike
β-crystallites for KE7 (26), we tentatively assume nonperiodic
stacking of slabs with a preferred slab orientation in each domain.
We observed strongly textured diffraction patterns in a zone where
the drying drop was arrested on the substrate before forming a residue.
Amyloid cross-β–structure formation has already been shown
for this zone for residues obtained by drying lysozyme solution drops
on a superhydrophobic substrate (27).Adiffraction pattern from the
interface zone, however, did not show the characteristic cross-
β–fiber pattern of ID3, LD6, and NL6, with the meridional and
equatorial directions at 90° but rather at ∼60° (Fig. 6A). We thus
exclude a structure based on an assembly of nanotubes as observed
for KE7 in acid solution (28) on the grounds of the lamellar morphology
(Fig. 6F) and suggest the model of lamellar stacking of
β-sheet slabs along the (b) chain axis (26). For the corresponding
unit-cell choice (26), we identified the strong meridional 200 reflection
(0.94 nm; H-bonding direction), 001 reflection (0.99 nm;
interchain packing), as well as the weaker 002 (0.48 nm) and 201
(0.43 nm) reflections (Fig. 6C). The equatorial reflections suggest
a period of 2.81 nm along the stacking direction (b axis) (Fig. 6B).
Based on the coincidence of the [100] and [001] directions, we
tentatively assume an intergrowth of slabs that are rotationally disordered
around the stacking direction. In view of a 3.03-nmb axis for
wild-type KE7 (26), the 2.81 nm value observed for the current KE7
with lamellar morphology suggests tilted 7-mer chains. For a possible
monoclinic structure with a unique c axis, the chains would be
tilted in the a/c plane by ∼30° from a normal to the a/b plane (Fig.
S4B). We cannot, however, exclude at present a triclinic unit cell.
The simulations of KE7 were carried out as before. All three
independent simulations of 50 ns resulted in similar structures.
Due to the high concentration and longer peptide length, the
peptides rapidly aggregated, forming a single cluster around 10
ns (Fig. S3D). The cluster was highly disorganized at this stage,
but remained stable throughout the simulations. At the end, KE7
formed a single large interconnected structure spanning the
length of the simulation box, contrary to the other peptides that
formed isolated fibers. Fig. 6D shows a snapshot of the final
structure from one KE7 simulation. The density maps of the final
structure were taken along the other two orthogonal planes (Fig.
S5 D and E). The density maps clearly show that KE7 forms
structures that are less dense and compact compared with the
other peptides. KE7 formed disordered aggregates that were
connected along all three axes, unlike the other peptides, which
assembled into isolated fiber-like structures along a single axis.
Discussion
The aim of this study was to compare the proposed self-assembly
mechanism for amyloid-like ultrasmall peptides with naturally
occurring amyloidogenic peptides. Interestingly, the self-assembly
of several natural core sequences proceeded via discrete stages similar
to the designed ultrasmall peptides, particularly with α-helical
structures as intermediates. This observation supports growing evidence
that early intermediates in amyloidosis are the toxic and
pathogenic species (10, 11). The toxicity of amyloidogenic peptides/
proteins is increasingly linked to their membrane permeabilization
and disruption capability (11, 29–31). The α-helical intermediates
observed in our study could play a key role, as α-helical structures are
known to facilitate membrane–peptide interaction and subsequent
membrane disruption (32). In addition, intermediates can promote
aggregation by increasing the local concentration of aggregating
species (31). This could occur by helix formation and helix–helix
association, or by creation of an anchor point via membrane–peptide
interactions for fiber assembly and elongation (3, 31, 33). In a diverse
cellular environment, conformational change of the amyloidogenic
species to an α-helical state could well be the trigger for self-assembly.
In fact, the work of Kelly and coworkers on transthyretin
suggests that induced conformational changes are sufficient to promote
spontaneous assembly of a functional protein into amyloid
fibrils (34). Previously, α-helical structures have been reported as key
kinetic intermediates for amyloid formation (31, 33). Here, we extend
this observation to small natural core sequences of four to seven
amino acids. These intermediates are observed by simply dissolving
the peptides in water, without the presence of helix-inducing compounds
such as hexafluoroisopropanol, membranes, or a hydrophobic
environment. Among the natural core sequences, DF5, with its
very stable α-helical intermediates, is the fastest to self-assemble into
fibrous aggregates. It is entirely conceivable that the conformational
transition from a random coil to an α-helical structure drives the
speed of self-assembly.
Similar Assembly for Amyloid Core Sequences and Aliphatic Peptides.
We have shown that naturally occurring NL6 (NFGAIL) and
DF5 (DFNKF), containing one or two aromatic residues, selfassemble
via the same mechanism as aliphatic peptides LD6 and
ID3. The final self-assembled structures formed by NL6, GA6, and
DF5 are morphologically and structurally similar to the designed
peptides. Our multimodal analysis of the amyloid peptide aggregates
clearly indicates that aromatic residues are not essential for
self-assembly. Nevertheless, they alter the kinetics of aggregation
as seen previously with the whole amyloid protein (35) and increase
the proportion of disordered aggregates. Molecular dynamics
simulations show that NL6 and KE7 form more disordered
aggregates compared with LD6 and ID3 in the initial stages. In
addition, NL6 takes longer to organize into an ordered fiber similar
to that ofLD6 and ID3.KE7 shows the fastest dynamics, with a single
cluster formed in 10 ns; however, the cluster is highly disordered and
loosely aggregated and a compact fiber is not seen within the simulated
time frame. Thus, aromatic and hydrophobic interactions
due to the phenylalanine residue shorten the lag phase for creation
of a nucleus, but do not lead to long-range fiber networks required
for hydrogelation. This is supported by our observation that KE7 is
the slowest to form a hydrogel among the 5- to 7-mers, whereasNL6
and DF5 form hydrogels with slight phase separation due to rapid
clumping of fibrous aggregates. Aliphatic LD6, on the other hand,
forms the stiffest and most homogeneous hydrogel.
Fig. 4. Molecular dynamics simulations in water and density maps of fibers.
(A−C) Final snapshots of the fibers from simulations of (A) 64 NL6, (B) 64 ID3,
and (C) 64 LD6 in water. Surprisingly, all three peptides form partially organized
fibers during the simulation.
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Amyloid-β’s KE7 Demonstrates Different Assembly. Examination of
KE7 containing the FF motif reveals very interesting results. First,
the KE7 aggregates do not exhibit the typical cross-β–diffraction
pattern that is used as a reference to distinguish amyloid structures
(4). KE7 assembles through a different mechanism not involving
α-helical intermediates. Lamellar β-sheet aggregates
made of flat nanotapes are formed, rather than helical nanofibers.
Flat nanotapes have been recently reported for KE7 capped
at both the N and C termini (36) but not with acetyl-KE7,
having a free C terminus. It is very likely that the FF motif has
a role in the unique behavior of KE7 under all examined aspects.
This is because the FF motif alone forms nanotubes and extremely
rigid structures rather than helical fibers (20, 37). Furthermore,
hydrogel formation and “amyloid-like” fibrils have only
been reported for FF with protecting groups such as Fmoc (20,
37). The steric hindrance introduced by FF might also prevent
formation of α-helical intermediates, ultimately leading to structurally
different aggregates (38).
Aromatic Residues Are Nonessential for Amyloid Self-Assembly. The
different behavior of KE7 is especially significant in view that its
FF motif has for long been proposed as the core motif for molecular
recognition and self-assembly of amyloid-β (15, 20).
Comparing two core sequences from Aβ, namely the fully aliphatic
GA6 and FF-containing KE7, raises several questions
pertaining to amyloidogenic potential and amyloid toxicity. Do
aromatic residues have an impact on the amyloidogenic potential
of a peptide? How can we accurately predict potent hotspots or
core sequences in an amyloid protein? Answering these questions
will help to better correlate in vitro data with in vivo toxicity
and pathogenicity. A recent investigation has unequivocally
identified fully aliphatic GA6 as the most potent hotspot for Aβ
aggregation (17). Thus, it is reasonable to infer from our results
that KE7 with the FF motif is not the most potent hotspot or core
recognition motif of Aβ.
In summary, we have proved that several natural core sequences
exhibit a similar mechanism of self-assembly as our rationally
designed ultrasmall peptides. Thus, it is justified to use the
aliphatic ultrasmall peptides as a simplified model system to study
amyloidosis. Using X-ray fiber diffraction, circular dichroism,
electron microscopy, rheology, gelation, and molecular dynamics
simulations to study different aspects of self-assembly, we have
shed light on the diverse factors that influence amyloid aggregation
and toxicity. We have shown that the core sequence KE7 of
amyloid-β, containing the FF motif, does not form a typical amyloid
cross-β structure and aggregates without the α-helical intermediate
step, giving a unique diffraction pattern and simulation
structure. Furthermore, our results indicate that aromatic interactions
may not be as crucial in amyloid formation as previously
postulated. More importantly, this study provides vital insight into
the nature of early intermediates, which will facilitate the search
for toxic species and potent hotspots of amyloid aggregation. It
also provides the basis for developing therapeutic drugs that
control and inhibit amyloid formation.
Materials and Methods
Peptides. Peptides with confirmed amino acid analysis (purity ≥95%) were
purchased from the American Peptide Company. Net peptide content varied
between 70% and 85%. All peptides were acetylated at the N terminus. Peptide
handling and hydrogel preparation were done as reported previously (12).
CD Spectroscopy. CD spectra were collected as reported previously (12).
FESEM and SEM Imaging. FESEM was performed as previously reported (12).
SEM images were recorded by a LEO 1530 Gemini SEM (Carl Zeiss SMT) at
20 kV. The SEM samples were sputtered with gold.
Fig. 5. CD spectra monitoring conformational transitions during self-assembly.
Random coil to α-helix to β-turn transition with increasing concentration
of (A) NL6 peptide from human amylin and (B) GA6 peptide from Aβ. (C)
Stable α-helical intermediates observed at different concentrations of DF5
from human calcitonin. (D) β-turn structure observed for DF5 at higher concentrations.
Fig. 6. X-ray fiber-diffraction pattern, simulation
snapshots, CD spectra, and SEM of KE7 peptide. (A)
Diffraction pattern of KE7 peptide with meridional
and equatorial directions defined as for a β-type
fiber-diffraction pattern. (B) Meridional intensity
profile fitted by four Gaussian profiles and a 0-order
polynomial. (C) Equatorial intensity profile by four
Gaussian profiles and a 0-order polynomial (d values
in nanometers). (D) Final snapshots of the structure
formed from the simulations of the 64 KE7 peptides
in water. The peptides form a larger interconnected
cluster instead of single fibers observed in the other
peptides. (E) CD spectra showing the direct conformational
transition of the KE7 peptides from random
coil to β-sheet with increasing concentration,
without going through the α-helical intermediate
step. (F) High-resolution SEM image of KE7 revealing
lamellar morphology.
Lakshmanan et al. PNAS Early Edition | 5 of 6
BIOPHYSICS AND
COMPUTATIONAL BIOLOGY
Rheology. Viscoelasticity of peptide hydrogels was measured using an ARESG2
rheometer (TA Instruments). A serrated stainless steel, parallel-plate
geometry of 8-mm diameter was used and the gap distance was maintained
between 0.8 and 2 mm. Oscillatory frequency sweep studies were performed
for a range of 0.1–100 rad/s, using either a 0.1% or 1% strain. Oscillatory
amplitude sweep studies were conducted from 0.1 to 100% strain with an
angular frequency of 1 rad/s. All measurements were done at 25 °C. The
ring-cast method used for hydrogel preparation is described in SI Materials
and Methods.
X-Ray–Diffraction Experiments. Aqueous solutions of 2.5 mg/mL for KE7 and
15 mg/mL for NL6 at pH 7 were prepared for X-ray fiber diffraction. Approximately
4-μL solution drops were deposited by pipette onto a superhydrophobic
poly(methyl methacrylate) surface and evaporated within
about 1 h under quasi–contact-free conditions into solid residues (21). Further
details are given in SI Materials and Methods.
Computer Simulations. Simulations of four peptides, namely LD6, ID3, NL6, and
KE7, were performed, using the Optimized Potentials for Liquid Simulations
(OPLS) force field. The extended simple point charge model (SPC/E) model
was used for water molecules and GROMACS version 4.0.5 was used for
molecular dynamics simulations, as reported (12). Further details are given in
SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Ulrich Hauser (Institute of Physics I,
University of Cologne) and Norma Greenfield (Robert Wood Johnson Medical
School) for helpful discussions, and Yihua Eva Loo and Michael Reithofer for
proofreading. X-ray–diffraction experiments were performed with the support
of staff handling the ID13 beamline, and SEM images were recorded by
I. Snigereva at the European Synchrotron Radiation Facility. Computing facilities
were provided by the A*STAR Computing Resource Center. This work is
funded by the Institute of Bioengineering and Nanotechnology (Biomedical
Research Council, Agency for Science, Technology and Research, Singapore)
and the Institute of High Performance Computing (Science and Engineering
Research Council, Agency for Science, Technology and Research, Singapore).
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6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1217742110 Lakshmanan et al.
Supporting Information
Lakshmanan et al. 10.1073/pnas.1217742110
SI Materials and Methods
Ring-Cast Method. For the rheology experiments, a ring-cast
method was used for hydrogel preparation to ensure uniform gel
volume and facilitate easy transfer of the intact gel onto the stage
for measurements. For comparison, a standard concentration of
12mMwas used for all peptides and measurements were taken 1–
2 d post gelation. After vortexing, 200-μL aliquots of the peptide
solution were immediately transferred into plastic ring casts of 9-
mm diameter and placed on clean 35-mm tissue-culture dishes.
The top and bottom of each ring cast was covered with a layer of
parafilm, and the tissue-culture dish was tightly sealed with
parafilm to prevent evaporation.
X-Ray Diffraction. Aqueous solutions of 2.5 mg/mL for KE7 and 15
mg/mL for NL6 at pH 7 were prepared for X-ray fiber diffraction.
Approximately 4-μL solution drops were deposited by pipette
onto a superhydrophobic poly(methyl methacrylate) surface and
evaporated within about 1 h under quasi–contact-free conditions
into solid residues (1). Raster-diffraction experiments (2) were
performed on the residues using a monochromatic beam of λ =
0.08122 nm, focused to a 200 (h) × 150 (v) nm2 spot with about 2 ×
109 photons/s flux using Si-refractive lenses in a crossed geometry
(3). A Maxipix detector (4), provided by the European Synchrotron
Radiation facility, with 512 × 512 pixels of 50 × 50 μm2 each was
used for data collection with ≤5 s per pattern.
Molecular Dynamics Simulation. All of the investigated peptides
(LD6, ID3, NL6, and KE7) have been described using the allatom
OPLS force field (5), and the water molecules were considered
explicitly using the SPC/E model (6). This force-field and
water-model combination has been found to give good results for
the hydration properties of amino acids (7). Molecular dynamics
simulations in the isothermal–isobaric ensemble were performed
using GROMACS version 4.0.5 (8) at time steps of 2 fs. Periodic
boundary conditions were applied in all three directions. Cutoff
radii were set at 0.9 nm for electrostatic interactions and 1.4 nm
for Lennard-Jones interactions. Long-range electrostatic interactions
were treated using the particle-mesh Ewald method (9).
Temperature coupling was achieved using the Berendsen thermostat,
but with an additional stochastic term to ensure a correct
kinetic energy distribution and produce a correct canonical ensemble
(10). Pressure coupling was achieved with the Berendsen
barostat (11). Relaxation times of 1 and 2 ps were used for the
thermostat and barostat, respectively.
The initial conformations of the peptides were obtained from
the final structure of a 50-ns molecular dynamics simulation of
a single molecule of each peptide in water at a temperature and
pressure of 298 K and 1 bar, respectively. This was to ensure
a relaxed starting conformation for the monomer before the
assembly process. Subsequently, 64 peptides were initially spaced
evenly within the simulation box and solvated with water. A high
concentration range was chosen so as to be able to observe fiber
formation within our simulation time. The concentrations of the
peptide solutions considered are given in Table S1. The box was
then subjected to an initial energy minimization to remove any
spurious overlaps before the molecular dynamics simulations
were performed on each system with the temperature coupled to
298 K and the pressure isotropically coupled to 1 bar. Three sets
of simulations were performed for each peptide and concentration
to establish the general validity of the results.
1. Accardo A, et al. (2010) In situ X-ray scattering studies of protein solution droplets
drying on micro- and nanopatterned superhydrophobic PMMA surfaces. Langmuir
26(18):15057–15064.
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condensed matter scattering and diffraction with microfocus techniques. Applications
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Heidelberg), Vol 776, pp 91–104.
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Phys Lett 87(12):124103-1–124103-3.
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with a synchrotron X-ray source. Nucl Instrum Methods Phys Res A 484(1-3):396–406.
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reparametrization of the OPLS-AA force field for proteins via comparison with
accurate quantum chemical calculations on peptides. J Phys Chem B 105(28):
6474–6487.
6. Berendsen HJC, Grigera JR, Straatsma TP (1987) The missing term in effective pair
potentials. J Phys Chem 91(24):6269–6271.
7. Hess B, van der Vegt NF (2006) Hydration thermodynamic properties of amino acid
analogues: A systematic comparison of biomolecular force fields and water models. J
Phys Chem B 110(35):17616–17626.
8. Hess B, Kutzner C, van der Spoel D, Lindahl E (2008) GROMACS 4: Algorithms for
highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory
Comput 4(3):435–447.
9. Essmann U, et al. (1995) A smooth particle mesh Ewald method. J Chem Phys 103(19):
8577–8593.
10. Bussi G, Donadio D, Parrinello M (2007) Canonical sampling through velocity
rescaling. J Chem Phys 126(1):014101.
11. Berendsen HJC, Postma JPM, van Gunsteren WF, Di Nola A, Haak JR (1984) Molecular
dynamics with coupling to an external bath. J Chem Phys 81(8):3684–3690.
Lakshmanan et al. www.pnas.org/cgi/content/short/1217742110 1 of 6
Fig. S1. Peptide hydrogels prepared by the ring-cast method and their rheological characterization. The molar concentration used for all peptides was 12 mM.
(A) LD6. (B) NL6. (C) KE7. (D) GA6. (E) DF5. (F) RADA. Images were taken 24 h after gel preparation, just before rheology measurements. (G) Frequency sweep
studies measuring loss modulus G′′ as a function of angular frequency ω. The trends are similar to those observed for the storage modulus (G′), with LD6 having
the highest and KE7 the lowest value. The G′′:G′ ratio (Tanδ) for all of the measured samples was much lower than 1, indicating dominance of the elastic solidlike
component over the viscous fluid-like component. (H) Amplitude sweep (G′′ vs. strain percent γ) shows a similar strain response for peptide hydrogels with
helical fiber networks and a different strain response for KE7 and RADA, which form lamellar β-sheets. KE7 and RADA alone show a temporary increase in the
loss modulus at a strain corresponding to the end of their linear viscoelastic range. The lamellar morphology and the viscous nature of the KE7 and RADA
hydrogels most likely lead to greater energy loss, especially around the yield point of the material.
A B
200 nm 200 nm
C D
Fig. S2. Morphological characterization of the peptide hydrogels using electron microscopy. (A) The scanning electron microscopy (SEM) image of NL6 attached
to a glass capillary showing a corrugated morphology. (B) SEM image of KE7 attached to a glass capillary showing a hollow shape. Lamellar layers can be
discerned even at this low magnification. (C) KE7 (12 mM) at a magnification of 60,000× showing flat nanotapes. (D) RADA (12 mM) at a magnification of
40,000× showing a flatlayer-like morphology.
Lakshmanan et al. www.pnas.org/cgi/content/short/1217742110 2 of 6
Fig. S3. Cluster size distribution as a function of time for the (A) 64 ID3, (B) 64 LD6, (C) 64 NL6, and (D) 64 KE7 peptides. All peptides form a single cluster within
30 ns, including the tripeptide ID3. KE7 showed the fastest dynamics, where a single aggregate was formed at around 10 ns. However, the aggregate was more
disordered and loosely connected compared with the other simulated peptides. LD6 formed a single stable cluster at around 12–13 ns, whereas NL6 formed
a single cluster at around 20 ns. ID3 was slowest to form a single cluster, at around 30 ns. This is as expected, because KE7 has the longest hydrophobic tail,
which increases the kinetics of aggregation, whereas ID3 has the shortest hydrophobic tail.
Lakshmanan et al. www.pnas.org/cgi/content/short/1217742110 3 of 6
Fig. S4. X-ray fiber diffraction and proposed structural model for KE7. (A) Composite diffraction image of bulk KE7 residue revealing two domains with
homogeneous β-reflection orientation. A fully extended 7-mer peptide chain at an angle of 30° to the fiber axis has a length of cos30°*(0.35 × 7) = 2.1 nm,
which is smaller than b = 2.81 nm [010]. This would agree with the KE7 raster diffraction. We performed raster diffraction on the bulk KE7 residue with 1-μm
step resolution. The composite diffraction image shown in Fig. S4A is based on the prominent 0.47-nm β-sheet reflection. The image reveals two domains with
preferred β-reflection texture. (B) Model of monoclinic slab with inclined 7-mer chains forming antiparallel β-sheets. The slabs are assumed to be rotationally
disordered along the stacking direction (c*). The structural model is based on β-sheet slabs stacked along the fiber axis (1). The diffraction pattern (Fig. 6A) does
not show the common orthogonal meridional and equatorial directions of a cross-β pattern but rather an angle of ∼60°, although the same types of reflections
are observed as for wild-type KE7 (1). The correlation length along the fiber axis is calculated as ∼3 nm from the width of the 010 reflection by Scherrer’s
equation (2), which corresponds roughly to the dimension of a single slab (1). We note that [100] and [001] directions coincide in the pattern and that the angle
of both directions with the [010] direction is ∼60° (Fig. 6A), suggesting tilted 7-mer chains. The tilting of the chains from a normal to the plane of the slabs is
deduced roughly from sin−1(2.81/3.03)∼70°. For a monoclinic lattice with unique c axis, the chains could be tilted in the a/c plane as shown schematically. A
triclinic space group can, however, not be excluded at present.
1. Inouye H, Gleason KA, Zhang D, Decatur SM, Kirschner DA (2010) Differential effects of Phe19 and Phe20 on fibril formation by amyloidogenic peptide A beta 16–22 (Ac-KLVFFAE-NH2).
Proteins 78(10):2306–2321.
2. Klug HP, Alexander LE (1974) X-Ray Diffraction Procedures for Polycrystalline and Amorphous Materials (Wiley Interscience, New York), 2nd Ed.
Fig. S5. Molecular dynamics simulations of 64 peptide molecules in water. (A–C) Density maps of the fibers taken along the plane perpendicular to fiber axis
for NL6, ID3, and LD6, respectively. This can give a rough estimate of the fiber dimensions. (D and E) Density maps of the final structure of KE7 taken along the y
and z axes, respectively. The cluster is seen to span the entire box length in all three directions.
Lakshmanan et al. www.pnas.org/cgi/content/short/1217742110 4 of 6
Movie S1. Simulation of 64 ID3 peptides in water. The formation of a fiber can be clearly observed.
Movie S1
Table S1. Concentrations of peptide solutions
Peptide Npep Nwat xpep mM
NL6 1 4,106 0.000243 13.28
64 15,748 0.00405 192.75
ID3 1 4,123 0.000242 13.28
64 9,074 0.007 338.0
LD6 1 4,108 0.000243 13.28
64 14,833 0.004296 207.57
KE7 1 4,095 0.000244 13.28
64 21,152 0.00302 145.78
Npep and Nwat are the number of peptide and water molecules in the
simulation box, respectively. xpep is the mole fraction of peptides. The concentrations
in mM are given in the last column.
Lakshmanan et al. www.pnas.org/cgi/content/short/1217742110 5 of 6
Movie S2. Simulation of 64 LD6 peptides in water. The formation of a fiber can be clearly observed.
Movie S2
Movie S3. Simulation of 64 NL6 peptides in water. The formation of a fiber can be clearly observed.
Movie S3
Lakshmanan et al. www.pnas.org/cgi/content/short/1217742110 6 of 6