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Chem. Sci., 2019, DOI: 10.1039/C8SC05771K.

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Cationic polymethacrylate-copolymer acts as an agonist for βamyloid and antagonist for amylin fibrillation•

Received 00th
January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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Bikash R. Sahooa, Takuya Genjoa, Takahiro W. Nakayama,b Andrea K. Stoddarda, Toshio Andob,
Kazuma Yasuharac, Carol A. Fierkea,d, Ayyalusamy Ramamoorthy*,a,e
In human, b-amyloid and islet amyloid polypeptide (IAPP, also known as amylin) aggregations are linked to Alzheimer’s
disease and type-2 Diabetes, respectively. There is significant interest in better understanding the aggregation process byusing chemical tools. Here, we show the ability of a cationic polymethacrylate-copolymer (PMAQA) to quickly induce bhairpin structure and accelerate the formation of amorphous aggregates of b-amyloid-1-40, whereas it constrains theconformational plasticity of amylin for several days and slow down its aggregation at substoichiometric polymer
concentrations. NMR experiments and microsecond scale atomistic molecular dynamics simulations reveal that PMAQA
interacts with b-amyloid-1-40’s residues spanning regions K16-V24 and A30-V40 followed by β-sheet induction. For amylin,
it binds strongly to the closest proximity of the amyloid core domain (NFGAIL) and restrains its structural rearrangement.
High-speed atomic force microscopy and transmission electron microscopy experiments show that PMAQA blocks thenucleation and fibrillation of amylin, whereas it induces the formation of amorphous aggregates of b-amyloid-1-40. Thus,
the reported study provides a valuable approach to develop polymer-based amyloid inhibitors to suppress the formation of
toxic intermediates of b-amyloid-1-40 and amylin.

Introduction
Self-assembly of amyloidogenic proteins is involved innumerous neurodegenerative diseases and other disease
including Alzheimer’s disease (AD) and Type-2 Diabetes (T2D).1,2
Nevertheless, our current understanding of the role of protein
aggregation in the pathogenesis of such diseases remains
elusive.3 Despite the recent advancements in high-throughput
screening of several anti-amyloidogenic compounds,4 there isno treatment for the protein aggregation based disorders
including AD and T2D.5 b-amyloid and islet amyloid polypeptide(amylin) aggregations are linked to AD and T2D, respectively.6
Both b-amyloid and amylin follow a conserved aggregation
pathway and form toxic oligomer intermediates. Thus, it has
been the subject of intense research to establish a pathologicalcorrelation and develop small molecules that interfere with theaggregation pathways of b-amyloid and amylin.6 The sequential
protein aggregation mechanism from a water-soluble monomer
to insoluble amyloid fibers triggering AD and T2D still remains
elusive. However, several studies have observed a conserved

a. Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI

48109-1055 (USA).
Frontier Research Center, Kanazawa University, Kanazawa 920-1192(JAPAN).
c. Graduate School of Materials Science, Nara Institute of Science and Technology,
Ikoma, Nara 6300192 (JAPAN).
d. Department of Chemistry, Texas A&M University, College Station, TX 77843 (USA).
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
b. Bio-AFM

pathway where both b-amyloid and amylin monomers
aggregate to form pre-fibrillar toxic oligomers followed bymatured b-sheet rich fiber structures.7 Thus, the sequential
aggregation products of b-amyloid or amylin have beentargeted to design potential inhibitors to interrupt amyloid
formation.4,8 Moreover, substantial research effort has been
devoted in developing strategies to reduce the formation of
toxic intermediates of b-amyloid or amylin. In response to this,several amyloid inhibitors or modulators have been clinicallytested for AD or T2D treatment. However, small molecule
inhibitors or modulators targeting amyloidosis have recently
faced several clinical trial challenges.8,9 In addition, thecomplexities associated with amyloid aggregation such as
biological pathways, involvement of other cellular key players,
structural heterogeneity etc. pose challenges to current
therapeutic developments.11 Among several compounds, thechemically conserved scaffold molecules characterized by multiaromatic groups have been tested in vitro10,11 or in vivo.14 These
compounds have been reported to modulate the amyloid
aggregation pathways. But the poor solubility andbioavailability affect their therapeutic nature and recently havebeen shown to be overcome using nanocarriers.15–17 Although,their mechanism of action remains unknown, covalent bonding,hydrogen bonding, hydrophobic interactions and π-π stacking
are thought to be the major driving forces for their inhibitory
mechanism of action.18–21 Oxidation of amyloid peptides bychemical compounds can also modulate amyloid aggregation.22
Despite substantial efforts, no significant new drugs have been
discovered against these most tenacious and unnerving medical
disorders. The successive failures of small molecule compounds

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direct researchers to develop new anti-amyloidogenic
molecules such as polymers, peptoids, macrocyclic peptides,
nanoparticles, molecular chaperones etc.8,23–27 Among them,several polymers characterized by their ionic properties havebeen tested to investigate their activities on amyloidogenic
aggregation. Notably, amine containing polymers, polyaminoacids, cationic surfactants and cellular polyamines have been
observed to modulate b-amyloid aggregation.28–30 Similarly,
controlled aggregation kinetics and toxicity of amylin using starpolymers and b-amyloid using polymer-nanodiscs have beenstudied recently19,20,25. Here we demonstrate the modulation ofamyloid aggregation pathways for amylin and b-amyloid-1-40
using a polymethacrylate derived copolymer (PMAQA) that has
been implicated in several biological studies including lipidnanodiscs formation, enhancement of drug-delivery,
modulating bioavailability and microencapsulation.31–33

Experimental section
Materials
The polymethacrylate quaternary ammonium copolymer(PMAQA, ~4.7 kDa) was synthesized and purified as reported
elsewhere.31 Unlabeled and uniform 15N isotope labeled full-lengthb-amyloid-1-40 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG
GVV) was recombinantly expressed in E. coli BL21 (DE3). The bamyloid-1-40 expression was followed from previously described
protocols 34,35 and purified by loading the samples to an ECOPLUS HPLC
column packed with the reversed-phase separation material.
Synthetic
human
amylin(KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH2) was purchased from AnaSpec at >95%
purity.
Sample preparation
The sample preparation was followed from the previously
described method.27 Briefly, 1 mg/mL b-amyloid-1-40 peptide wasdissolved in 5% (v/v) NH4OH and aliquoted to 0.1 mg/mL followed bylyophilization. The b-amyloid-1-40 peptide powder was resuspended in 10 mM sodium phosphate, pH 7.4 and sonicated for 30s followed by centrifugation at 14,000 ´ g for 15 min at 4 °C toremove small aggregates. 1 mg/mL amylin was treated with
1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and kept on ice for 30
minutes. The peptide solutions were aliquoted to 0.1 mg/mL andlyophilized. The amylin powder was re-suspended in 30 mM sodium
acetate buffer, pH 5.5 and sonicated for 30s. The PMAQA powder (10mg/mL) was dissolved in deionized water.
Thioflavin-T fluorescence assay
Thioflavin T (ThT) fluorescence assays were performed tomonitor the aggregation kinetics of 5 µM b-amyloid-1-40 or amylinat 37 °C in the presence of PMAQA (0.1, 0.2, 1, 2.5, 5, 6, 7.5, 10, and50 µM) and 10 µM ThT. Fisher 96-well polystyrene plates with asample volume of 100 μL/well in triplicate were used for the ThT
assay. The kinetics of amyloid formation was monitored at 3-minintervals with no-shaking conditions for 4 days using a microplate
reader (Biotek Synergy 2) with an excitation and emission
wavelengths of 440 and 485 nm, respectively.
Circular dichroism and Fourier transform-infrared spectroscopy
b-amyloid-1-40 or amylin secondary structural transition in thepresence or absence of PMAQA was studied by Far-UV circulardichroism (CD) using a JASCO (J820) spectropolarimeter.
A 1Onlinemm
View Article
DOI: 10.1039/C8SC05771K
light-path length cuvette containing 25 µM b-amyloid-1-40
or amylin
solution titrated with an increasing concentration of PMAQA(ranging from 0.25 to 50 µM) was used to monitor the evolution of
structural transition at 25 °C. The samples (peptide:polymer=1:1.5)
were stored at room temperature and the CD spectra were recorded
for 5 days at different time intervals. The CD spectra were averaged
and conveyed as the mean residue ellipticity [Q] after subtracting thesignal from a solution without peptide. Fourier transform-infrared(FT-IR) spectra were measured for 25 µM b-amyloid-1-40 or amylin
mixed with 35 µM of PMAQA (incubated for 6 hours followed bylyophilization) in transmission mode within a range of 4000–400 cm−1
using a Thermos scientific ATR-FTIR instrument. 10 µM of β-amyloid1-40 or amylin was incubated with 15 µM of PMAQA for ~5 minutes
and overnight, respectively, for size distribution analysis. The
resultant solutions were purified using size exclusion
chromatography (SEC) by passing through a superdex 200 Increase
300/10 GL column operated on an AKTA purifier (GE Healthcare,
Freiburg, Germany).
NMR
1D and 2D NMR spectra were recorded on a 600 MHz Bruker
Avance III NMR spectrometer equipped with a z-axis gradientcryogenic probe. Unlabeled amylin (50 µM) dissolved in 30 mM
sodium acetate, pH 5.5, and unlabeled β-amyloid-1-40 or uniformly
15N-labeled β-amyloid-1-40 peptides (60 µM) dissolved in 10 mM
sodium phosphate, pH 7.4, buffer containing 90% H2O/10% 2H2O wasused for NMR measurements. The 2D 15N/1H SOFAST-HMQC36 NMR
titration experiments of β-amyloid-1-40 (60 µM) with 0.6, 1.2 and 3µM PMAQA were recorded at 10 °C with 64 scans and 200 t1
increments. The NMR spectra were processed using TopSpin 3.5(Bruker) and analyzed using Sparky.37
High-speed AFM and Transmission Electron Microscopy imaging
A high-speed atomic force microscopy (HS-AFM)
instrument 38,39 was operated at the tapping mode with a smallcantilever (BL-AC10DS-A2, Olympus with spring constant k = 0.1
N/m, and resonance frequency f = ~400 kHz in water). Eachcantilever has an amorphous carbon tip at its top. The tip was
prepared by electron beam deposition with a scanning electron
microscope (ELS-7500, Elionix). The free oscillation amplitude of
the cantilever and the typical set-point were ~1 nm and 80% of thefree oscillation amplitude. For the HS-AFM sample stage, a freshly
cleaved mica disc (of 1 mm in diameter) fixed on a glass rod (of 2
mm in diameter) was used. Before the deposition of amylin fibril
seed, each mica disc was freshly cleaved. For 3aminopropyltriethoxysilane (APTES)-modification, mica was
immersed with 2 µL of ten-thousand-fold dilution of APTES withwater followed by the removal of unbound APTES with 60 µL ofwater. Amylin fibril seeds for the imaging were prepared byincubation of 60-100 µL of 5 µM amylin in 30 mM sodium acetate,
pH 5.5 for 2-3 days followed by sonication with a handheld
sonicator (UR-21P, TOMY). Validation of the sonication condition
was checked from AFM images (typical sonication condition: tentimes of ~0.5 s at the output level 3). 2 µL of fibril seeds was
deposited on bare mica or APTES-modified mica followed byincubation for about 5 minutes. After the removal of unboundseeds with 20 µL of 30 mM sodium acetate, pH 5.5, the scannerwith the sample stage was set on the scanner holder so that thestage was immersed in 60 µL of 30 mM sodium acetate, pH 5.5, inthe sample chamber with the cantilever. After the observation of
the fibril seeds, the buffer solution in the chamber was replaced

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with 5 µM amylin with/without equimolar PMAQA. HS-AFM
observation was started just after the replacement of the sample
chamber solution. The HS-AFM movie analysis and kymographs
were prepared using ImageJ (NIH) plugin as described elsewhere.40
5 µM of β-amyloid-1-40 monomers dissolved in 10 mM sodium
phosphate, pH 7.4 or 5 µM of amylin monomers dissolved in 30 mM
sodium acetate, pH 5.5, were incubated for 24 hours undercontinuous agitation with/without equimolar concentration of
PMAQA. β-amyloid-1-40 or amylin fibers were prepared byincubating 5 µM of peptide monomers in respective buffer as
described above at room temperature for 3 days under continuous
agitation. Then, the fibers were sonicated using an ultrasonic bathsonicator for 2 hours at room temperature to generate short fibers,
which were used as amyloid seeds. The sonicated fibers wereincubated with 5 µM of PMAQA in respective peptide buffers at room
temperature for 24 hours under continuous agitation to check theeffect of polymer on fiber morphology. In addition, to monitor
PMAQA’s effect on the self-seeding reaction, sample solutions
containing sonicated fibers were incubated with 5 µM monomers ofrespective peptides for 24 hours in the presence or absence of 5 µM
PMAQA. 10 μL of all sample solutions described above were added
to a collodion-coated copper grid and incubated for 3 minutes atroom temperature followed by three times rinsing with double
deionized water. Then, the copper grid was stained with 3 μL of 2%(w/w) uranyl acetate and incubated for 2 minutes followed by threetimes rinsing with double deionized water. The sample grids weredried overnight under vacuum and used to obtain transmission
electron microscopy (TEM) images using a HITACHI H-7650
transmission microscope (Hitachi, Tokyo, Japan) at 25 °C.
MD simulations
The 2D structure of PMAQA (Fig. S1) was created using
ChemDraw 16.0 and exported to Chem3D for energy
minimization. The 3D structure of PMAQA and its topology files
were created using ATB builder41 from the energy minimized 2D
structure for all-atom molecular dynamics (MD) simulation. The
solution NMR structures of β-amyloid-1-4042 (PDB ID: 2LFM)
and amylin43 (PDB ID: 5MGQ) were considered as the initialstructures for PMAQA interaction analysis; the amino acidsequences of the peptides are shown in Fig. S1. The MD system
was built in GROMACS44 software package, version 5.0.7(GROMOS96 54A745 force field), by placing β-amyloid-1-40 or
amylin at the center of a cubic box and the polymer ~ 1 nm awayfrom the protein. The MD systems were solvated using SPC/E
water (≈ 1000 kg m-3) and neutralized by adding counter ionsfollowed by energy-minimization using the steepest-descentmethod. Short NVT (100 ps) followed by 1 ns NPT (310 K and 1bar) was performed to equilibrate the MD systems. MD
simulations were carried out using 3D periodic boundary
conditions over a production run of 0.7 and 1µs for amylinPMAQA and β-amyloid-PMAQA systems, respectively. MD
trajectories were interpreted using visual molecular dynamics46
and images were built using Discovery studio visualizer 3.5. The
binding free energy was calculated for amylin-PMAQA and βamyloid-PMAQA complexes using MM/PBSA by retrieving 500
structures from the last 100 ns MD simulation.47 The GMXAPBS
tool was used for the free energy calculation as described
elsewhere.48–50

Results and discussion

View Article Online

DOI: 10.1039/C8SC05771K
The effect of PMAQA on β-amyloid-1-40 andamylin aggregation
kinetics was monitored using ThT based fluorescence assay. ThT
aggregation assays monitored for 4 days showed opposite activities
for β-amyloid-1-40 and amylin aggregation in the presence of
PMAQA (Figs. 1 and S2). As shown in Fig. 1 (a and b), PMAQA
accelerated β-amyloid-1-40 and slowed down amylin aggregations.
At a low polymer concentration, i.e. peptide:PMAQA molar ratio of
1:0.02 or 1:0.04, a significant difference in the lag-times of βamyloid-1-40 and amylin aggregation was ascertained (Fig. 1a and b).
In the presence of 0.02 µM of PMAQA, the β-amyloid-1-40 fibrillation
growth was observed after two days (blue) as compared to theaggregation after three days for the control sample (red). A slightincrease in the polymer concentration, i.e. 0.04 µM of PMAQA(green), significantly accelerated β-amyloid-1-40 fibrillation with anincreased ThT fluorescence intensity (Figs. 1a and S2a). While further
increase in the polymer concentration to 1:0.2 or 1:0.5 βamyloid:PMAQA molar ratio also accelerated the peptide
aggregation. But, a relatively lower ThT intensity was observed for
1:0.5 as compared to that obtained for 1:0.2 (or 1:0.04). As shown in
Fig.1a, the observed ThT intensities are higher for all thesubstoichiometric concentrations of PMAQA than the control (i.e., in
the absence of PMAQA). At equimolar or superstoichiometricconcentration of PMAQA (Figs. 1a and S2b), β-amyloid-1-40
fibrillation was ≈6 times faster than that observed for the control
sample.
Unlike the effect of PMAQA on β-amyloid-1-40 fibrillation, amylin
aggregation was slowed down at substoichiometric PMAQA
concentration, i.e. amylin:PMAQA molar ratio of 1:0.02 or 1:0.04 (Fig.
S2c). Increasing the polymer concentration to 1:0.2 amylin:PMAQA
molar ratio further slowed down the aggregation with a lag-time over
2 days as compared to its aggregation in ~1 day in the absence of
PMAQA (Fig. 1b). Further increase in PMAQA concentration, i.e. at
equimolar polymer:peptide or superstoichiometric PMAQA
concentrations, amylin aggregation was significantly slowed down as
revealed from the negligibly small ThT fluorescence intensity (Figs.
1b and S2d). The observed increase in the ThT intensity in both
peptidesatsubstoichiometric
PMAQA
concentrations(peptide:polymer 1:0.5 or lower) indicates the presence of a highquantity of β-amyloid or amylin fibers. On the other hand, atsuperstoichiometric PMAQA concentrations (peptide:polymer 1:1 orhigher), a substantial decrease in the ThT intensity indicates thepresence of less amount of fibers. Specifically, the low ThT intensity
observed for β-amyloid-1-40 could also indicate the formation ofFig. 1 Effect of PMAQA on the aggregation kinetics of β-amyloid-1-40 or
amylin. Relative ThT fluorescence of 5 µM β-amyloid-1-40 in 10 mM
sodium phosphate, pH 7.4 (a), or amylin in 30 mM sodium acetate, pH 5.5(b) in the presence of variable PMAQA concentrations at the indicatedpeptide to polymer molar ratios. The data in both graphs represent the
average of ThT values performed in triplicate as shown in Figure S2.

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amyloid:PMAQA molar ratio (Fig. 2a). A further increasein PMAQA
View Article
Online
DOI: 10.1039/C8SC05771K
concentration to 1:1 β-amyloid:PMAQA molar
ratio marginally
increased the β-sheet contents in β-amyloid-1-40 (7.7/28.2% of α/βcontent). The shift of CD minima at ≈200 nm and decrease in [Q] withan increasing concentration of PMAQA indicated its activity on
accelerating β-amyloid-1-40’s aggregation as observed in the ThT
assay (Fig. 1a). Remarkably, unlike β-amyloid-1-40, no significant
conformational changes were observed in amylin when titrated with
0.2 to 2 equivalents of PMAQA with a CD minimum centered at ≈200
nm with a very little change in [Q]that represents a partially folded
random-coil rich conformation (Fig. 2b).
Table 1 Secondary structure assessment (%) of amylin from CD spectra (Fig.
2d and f) in the absence or presence of PMAQA by BESTSEL.52

Time
0d
1d
2d
3d
4d
Amylin in 30 mM sodium acetate, pH 5.5
α-helix
0.7
1.4
0
0
1.3
42.1
37.5
54.0
59.0
53.1
bz
0
3.9
0
0
0.2
by
Amylin+PMAQA in 30 mM sodium acetate, pH 5.5
α-helix
14.5
9.1
10.6
5.3
5.3
21.7
29.7
28.1
30.3
33.4
bz
0
0
0
5.7
3.7
by

5d
0.4
53.0
0
6.6
25.4
13.4

z: Antiparallel b-sheet; y: Parallel b-sheet

Fig. 2 Conformational analysis of β-amyloid-1-40 and amylin with andwithout PMAQA. Far-UV CD measurements monitoring the structuralchanges in 25 µM of β-amyloid-1-40 in 10 mM sodium phosphate, pH
7.4 (a) and in 25 µM of amylin in 30 mM sodium acetate, pH 5.5 (b) in
the presence of PMAQA incubated for ~10 minutes at room
temperature at the indicated peptide:PMAQA molar ratios (a and b).
Time-lapsed structural changes in 25 µM β-amyloid-1-40 (c and e) andamylin (d and f) in the absence (c and d) and presence (e and f) of 35 µM
PMAQA; time intervals used for results presented in c-f are indicated in(c). FT-IR spectra of β-amyloid-1-40 (g) and amylin (h) in the presence(green) and absence (red) of PMAQA obtained after 6 hours; peptide
concentration and buffer conditions were as described for the samples
used in CD measurements. Changes in β-amyloid-1-40’s secondary
structure are highlighted in yellow in the fingerprint regions (1550-1725
cm-1) (g, top).

Next, the conformational changes in both peptides in the presence
and absence of PMAQA were investigated using far-UV CD
experiments. The CD spectra revealed a gradual structural transition(from unfolded to folded) in β-amyloid-1-40 when titrated with
1:0.01 or 1:0.04 β-amyloid:PMAQA molar ratio as indicated by areduction in CD molar ellipticity [Q] and a small change in CD minimaat ≈200 nm (Fig. 2a). The reduction in [Q] indicates β-amyloid-1-40
aggregation and is in line with the observed ThT aggregation (Fig. 1a).
A partial helical CD spectrum containing 9.4/26.1% of α/β secondary
contents (as estimated by BESTSEL52) was observed at 1:0.2 β-

We next monitored the stability of the PMAQA induced βamyloid-1-40 or amylin secondary structures for 5 days by incubating
the peptides at room temperature in the absence or presence of 1.5
molar equivalents of PMAQA (Fig. 2c-f). The time-lapse CD spectra of
β-amyloid-1-40 or amylin in the absence of PMAQA showed asequential structural transition from a random-coil rich (negative
peak ≈200 nm) to β-sheet structure (Fig. 2c and d). While b-amyloid1-40 in solution are reported to exhibit a sequential structural change
to form cross b-sheet structures over several days (Fig. 2c),53 in thepresence of 1.5 molar equivalents of PMAQA a rapid b-sheet
induction within several minutes was observed (Fig. 2e). The
observed rapid structural change correlates with the ThT aggregation
kinetics (Fig.1a) and indicates a reduction in the level of potential βamyloid-1-40 oligomers that are reported to be neurotoxic.53,54
Similar to the amyloidogenic property of β-amyloid-1-40, amylinin solution after 2 days depicted a CD spectrum with a maximum at≈200 and a minimum at ≈218 nm indicating its transitory statecharacterized by an increasing percentage of β-sheet (54%) (Fig. 2d).
But, unlike β-amyloid-1-40, and as observed in ThT assays for amylin(Fig.1b), CD spectra showed a relatively slow change in amylin’s
secondary structure when incubated with 1.5 molar excess of
PMAQA. The CD minimum was observed at ≈200 nm up to day-3 andthe spectra were quantified using BESTSEL (Fig.2f, Table 1).
Secondary structure assessment of PMAQA bound amylin from CD
spectra showed a relative increase in α-helix (14.5%) and decrease inβ-sheet (21.7%) content as compared to that observed in theabsence of PMAQA (Table 1). In addition, an increased percentage ofparallel β-sheets (13.4 % on day-5) was observed for amylin in thepresence of PMAQA over time as compared to 0% (on day-5) in theabsence of PMAQA. This observation indicates that PMAQA boundamylin could have both anti-parallel and parallel β-structures. Suchobservations have been found previously using X-ray crystallography
in different segments of amylin (segment 13-18 with parallel andanti-parallel β-structures for segments 16-21, 22-28, and 23-29).55
On the other hand, β-amyloid-1-40 showing a positive at ≈200 and
negative at ≈225 nm CD bands in the presence of PMAQA indicatesthe formation of a predominant β-sheet structure (Fig.2e). The
observed CD minimum with a red-shift (from ≈222 on day-1 to ≈225

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morphologically distinct fibers, i.e. amorphous like aggregates that
depict low ThT fluorescence intensity.51

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ARTICLEnm on day-5) in β-amyloid-1-40 correlates to previously observed βsheet rich supramolecular structures in modified β-amyloid-1-40 andother small peptide aggregates.56,57
Next, we carried out FT-IR experiments on samples after 6 hours
of incubation under similar experimental conditions used for CD
measurements to further confirm the observations from CD
experiments. As shown by the FT-IR spectra in Fig.2g, predominant
β-sheet structures of β-amyloid-1-40 were observed as indicated bya sharp amide I peak at 1628 cm-1 along with an increasing
percentage of turns (at 1675 cm-1) in the presence of PMAQA, which
are in qualitative agreement with CD results shown in Fig.2e. In thecase of amylin, FT-IR spectra showed a minor change in thecharacteristic of the helical band at 1650 cm-1 in the presence of
PMAQA (Fig. 2h). This indicates that PMAQA binding restrains theconformation of partially folded amylin43. The β-sheet rich β-amyloid
and partially folded amylin conformations in the presence of PMAQA(Fig. 2) coupled with ThT observations (Fig. 1) indicated the presenceof distinct peptide species such as fibers or low-order aggregates. To
probe this, SEC profiling was performed to quantify the size
distribution of the PMAQA bound peptide species. SEC analysis of βamyloid-1-40 incubated with 1.5 molar excess of PMAQA for ~5minutes at room temperature showed two different elution profiles.
The fractions collected at ~5 to 12 mL correspond to amyloid fibers
or protofibers and that collected at ~20 to 25 mL are free polymers
or low-order or monomeric β-amyloid-1-40 (Fig. S3a).58 Remarkably,
SEC profile for PMAQA-amylin mixed solution, incubated for a
relatively longer time (overnight) at room temperature, exhibited
low-order or monomeric amylin and free polymers eluted at ≈20 to
25 mL (Fig. S3b).59 Taken together, the above described experimental

results present a counter active role of PMAQA on b-amyloid-1-40
View Article Online
DOI: 10.1039/C8SC05771K
and amylin aggregation.
Next, we studied the binding mechanism of PMAQA with βamyloid-1-40 or amylin using an integrated NMR and MD simulation
approach. The amide-NH region of 1H NMR spectra obtained in theabsence of PMAQA showed a monomer or low-order aggregates forboth b-amyloid-1-40 and amylin as indicated by the dispersed NMR
peaks (Fig. 3). A substantial change in 1H NMR spectra of β-amyloid1-40 was observed at 1:0.02 b-amyloid:PMAQA molar ratio (Fig. 3,
pink trace). Remarkably, the loss of amide-1H peaks of β-amyloid-140 was observed when the PMAQA concentration (1:0.05 bamyloid:PMAQA ratio) was slightly increased (Fig. 4d). This agrees
with the PMAQA induced β-amyloid-1-40 aggregation observed in
ThT assays. In contrast, the amide peaks of amylin were observed
even when titrated with 1.2 molar equivalent of PMAQA (Fig. 3, blue
trace). These NMR findings are in good agreement with the observed
conformational transition from CD and ThT based aggregation results(Figs. 1b and 2b). Interestingly, a significant line broadening observed
for the proton peak of PMAQA’s –NR3+ group (at 2.97 ppm) in thepresence of b-amyloid-1-40 which indicates the interaction of –NR3+
with b-amyloid-1-40. In contrast, a sharp proton peak for PMAQA’s –NR3+ was observed in amylin solution (Fig.3). This observation most
likely indicates the formation of an electrostatic interaction between
the cationic PMAQA and anionic β-amyloid-1-40, as β-amyloid-1-40(at pH=7.4) and amylin (at pH=5.5) carry negative (-3) and positive(+4) charges, respectively.60,61 Thus, while the cationic group of
PMAQA binds strongly to anionic β-amyloid-1-40, a repulsive force
could be expected in the presence of cationic amylin.

Fig. 4 SOFAST-HMQC NMR titration of PMAQA with β-amyloid-1-40. 2D
1H/15N SOFAST-HMQC NMR spectra for β-amyloid-1-40 (60 µM)
dissolved in 10 mM sodium phosphate, pH 7.4, (a) in the absence (red)or (b-d) presence of PMAQA (blue) at the indicated concentration (top).
NMR spectra were recorded at 10 ̊C on a 600 MHz Bruker NMR
spectrometer. Blue contours are presented with 1.5 times higher values
as compared to red contours.

Fig. 3 1H NMR spectra of β-amyloid-1-40 or amylin. NMR spectra showing
the interaction of PMAQA with β-amyloid-1-40 (60 µM β-amyloid-1-40, 1.2µM PMAQA dissolved in 10 mM sodium phosphate, pH 7.4) or amylin (50µM amylin, 60 µM PMAQA dissolved in 30 mM sodium acetate, pH 5.5). Thechange in the NMR signal intensity of the –NR3+ proton in PMAQA or protein
amide region (H-N) is highlighted.

To gain further atomistic insight into the mechanism, β-amyloid1-40 aggregation was monitored using 2D 15N/1H SOFAST-HMQC
experiment at variable substoichiometric polymer concentration;
since 1H NMR of amylin showed very little change in the amide region
at superstoichiometric PMAQA concentration, further NMR
experiments were not carried out on amylin. As shown in Fig. 4a-d,
intensity reduction and chemical shift changes were observed for βamyloid-1-40 residues indicating a PMAQA induced structural
rearrangement for β-amyloid-1-40. A substantial loss of signal
intensity was observed in β-amyloid-1-40 spanning regions K16-V24
and A30-V40 (as per previously published assignments62) indicates apotential site of PMAQA’s interaction with β-amyloid-1-40 at 1:0.01

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and 1:0.02 β-amyloid:PMAQA molar ratio (Fig. 4b and c). A further
increase in PMAQA concentration (1:0.05 β-amyloid:PMAQA molar
ratio) increased the line-broadening and also resulted in the loss of
15N/1H resonances (Fig. 4d). This indicates that PMAQA interaction

PMAQA (Fig. S4a). Amylin residues such as Asn21,View
Ile26,
ArticleSer28,
Online
DOI: 10.1039/C8SC05771K
Thr30, Asn31, Ser34 and Tyr37 were identified
to be involved inintermolecular hydrogen bonding interactions with PMAQA (Fig.5b,
Table S2). Overall, PMAQA exhibited a greater number of hydrogenbonds with amylin than with β-amyloid-1-40 indicating a relatively
stronger binding affinity of PMAQA to amylin (Fig. S4a).
Computation of binding energy using MM/PBSA presented
distinct energetic parameters that favor PMAQA binding to βamyloid-1-40 or amylin. The binding energy estimated for PMAQA-βamyloid-1-40 and PMAQA-amylin complexes were -16.5±10.1 and 27.4±11.7 kcal mol-1, respectively. The energetic parameter analysis
shown in Table 2 indicates that coulombic and van der Waals
interactions play a crucial role in PMAQA binding to β-amyloid-1-40,
while the polar solvation energy restricts the interaction. On theother hand, PMAQA binding to amylin is favored by the nonpolar
component of the solvation free energy and van der Waals
interaction, while columbic interaction disfavors the complex
formation (Table 2). This indicates that the opposite charges in βamyloid-1-40 and amylin are the major factors for their distinguishedamyloid aggregation behavior in the presence of PMAQA. The
electrostatic interaction or repulsion in β-amyloid-1-40 or amylin
interaction with PMAQA, respectively, could contribute to theacceleration or deceleration of amyloid aggregation as observed inprevious studies using differently charged nanoparticles.63,64
Table 2. MM/PBSA based binding free energy (kcal mol-1) calculation.
Polar Contribution
1ΔG
2ΔG
bind
coul

Fig. 5 Structural insights into the PMAQA interaction with b-amyloid-140 or amylin. MD snapshots showing PMAQA (shown in ball and sticks)
interaction with b-amyloid-1-40 (a) or amylin (b) shown as cartoon. The
PMAQA binding peptide amino acids (shown in sticks) are labelled andhydrogen bonds are shown in black dashed lines in Discovery Studio
Visualizer. (c) Peak intensities measured from 2D SOFAST-HMQC spectra(Figure 4) of 60 µM b-amyloid-1-40 in the absence or presence of 0.6 µM
PMAQA. The yellow area highlights the b-amyloid-1-40‘s residues withsignificantly reduced signal intensities. (d) Root mean square fluctuation(RMSF) of residues in b-amyloid-1-40 (grey) or amylin (red) interactingwith PMAQA derived from 1 or 0.7 µs MD simulations, respectively. The
yellow region indicates a comparatively flexible b-amyloid-1-40 Nterminal domain. The blue and orange regions indicate aggregation core
domains of amylin and b-amyloid-1-40, respectively. The average RMSF
value is shown using dashed horizontal lines.

with β-amyloid-1-40 induces the aggregation of the peptide.
To further explore the binding mechanism of PMAQA with amylinor β-amyloid-1-40 at structural level, we performed all-atom MD
simulation on a time scale of 0.7 or 1 µs, respectively. Structural
analysis showed a substantial number of hydrogen bond formation
between PMAQA and β-amyloid-1-40 over a time-scale of 1 µs (Fig.
S4a). All-atom MD simulation revealed potential electrostaticinteractions or hydrogen bonds between the Glu22 and Asp23
residues of β-amyloid-1-40 and the polymer’s –NR3+ (Fig. 5a),whereas no interaction with β-amyloid-1-40’s charged residues in
the N-terminal (Asp1, Glu3 and Asp7) were observed. Other residues
such as Leu17, Phe20, Val24, Ser26, Asn27, Ile31, and Ile32 wereobserved to interact with PMAQA through hydrogen bonding orhydrophobic interactions (Fig 5a, Table S1). This correlates with the
NMR results that showed substantial decrease in the NMR signal
intensity in β-amyloid-1-40 regions spanning K16-V24 and A30-V40(Fig. 5c). MD calculations also revealed several intermolecularhydrogen bond and hydrophobic interactions between amylin and

3ΔG

ps

Non-Polar Contribution
5ΔG
6ΔG
po
vdw
nps

4ΔG

7ΔG

np

β-amyloid+PMAQA

-16.5
±10.1

-521.4±132.7560.0±137.0

38.5

-48.0
±7.2

-7.0
±0.8

-55.1

amylin+
PMAQA

-27.4
±11.7

84.9
±106.2-50.0±103.6

34.8

-54.7
±7.8

-7.6
±0.9

-62.3

1Binding free energy, 2Coulombic term, 3Polar solvation, 4Polar contribution, 5van der
Waals energy, 6Nonpolar solvation, 7Nonpolar contribution.

The root mean square deviation (RMSD) of the backbone atoms
calculated from 1 µs MD simulation of β-amyloid-PMAQA systemshowed an RMSD plateau with an average value ≈11 Å (Fig. S4b). A
relatively small backbone RMSD (average value ≈8.8 Å) was observed
for amylin-PMAQA system indicating a comparatively stable complex
formation (Fig. S4c). The RMSD of PMAQA calculated from all-atoms
depicted a nearly equal RMSD value ≈6.5 Å for both MD systems (Fig.
S4d). Overall, the stable peptide backbone with an average RMSD <2 Å over several hundreds of nanoseconds indicate a strong coupling
between PMAQA and amylin or β-amyloid-1-40 over the ~µs timescale MD simulation. Further the root mean square fluctuation(RMSF) analysis of individual amino acids in β-amyloid-1-40 or amylin
highlighted the potential PMAQA interaction regions. The amyloid
aggregation core domains in both β-amyloid-1-40 (KLVFF) and amylin(NFGAIL) depicted an RMSF value lower than their corresponding
average values as indicated in Fig. 5d. In amylin, residues 7-16
showed the lowest RMSF values that folded to a stable α-helicalconformation. This indicates the PMAQA interaction restrain the
structural and dynamic properties of amylin. The tight coupling of
PMAQA close to the proximity of NFGAIL region in amylin followedby restriction in protein structural rearrangement reveal mechanistic
insights into PMAQA’s antagonist property in amylin aggregation.
Further, the secondary structure analysis evolved from MD
trajectories in amylin-PMAQA complex showed no significant
secondary structural change during the MD simulation in amylin (Fig.
S5, bottom). On the other hand, a substantial secondary structurechange was observed in β-amyloid-1-40 complexed with PMAQA
including the induction of an antiparallel β-sheet along the terminal

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residues (Figs. 5a and S5, top). The interaction of PMAQA with thecentrally located residues of β-amyloid-1-40 such as Glu22, Asp23,
Val24, Ser26 and Asn27 induced a random-coil and helix
conformation in β-amyloid-1-40. Structural changes in β-amyloid-140 by affecting these central residues by mutation, disruption of the
D23-K28 salt-bridge or binding of hexapeptide of genetic β-amyloid1-40 variants has been reported previously to be crucial inaccelerating β-amyloid-1-40 aggregation and induction of terminal βstructure.65–68 The charge-charge interaction between PMAQA (NR3+) and Glu22/Asp23 in β-amyloid-1-40 could interfere with theearly oligomer morphology and distribution of large β-amyloid-1-40
aggregates as observed in SEC (Fig. S3).69 The binding of PMAQA to
Glu22 and Val24 also affects the hydrophobic interaction between
Val24 and Lys28 and electrostatic interaction between Lys28 and
Glu22/Asp23.70
We next monitored the aggregation kinetics of amylin on real-

6b). These observations agree with the reported observationsforView Article Online
71,72 Remarkably,
DOI: 10.1039/C8SC05771K
other amyloidogenic peptides including β-amyloid.
in the presence of equimolar PMAQA, both amylin fibril growth andde novo nucleation were found to be blocked (Fig. 6c, Movie S2). In
addition, no significant difference in the fiber length and population
were observed between 0 s and 1350 s (Fig. 6c). Kymographs ofamylin fibrillation in the presence of PMAQA showed no growth andindicates the polymer blocks the recruitment of amylin monomers to
the fiber end to proceed the seeding reaction (Fig. 6d). These distinct
morphological features observed on both bare mica and APTES-micasuggest that the electrostatic interaction between the sample andthe stage did not affect amylin’s aggregation and the interactions
with PMAQA. The HS-AFM experimental results, therefore, revealed

Fig. 6 Real-time monitoring amylin fibrillation using HS-AFM. HS-AFM
images show seeding reaction (fibril growth) of 5 µM amylin monomers
in the absence (a) or presence (c) of equimolar PMAQA. These images
were extracted at the indicated time intervals from movies S1 and S2(included in the Supporting Information). The growth of fibers as afunction of time are indicated by arrows. The de novo nucleated amylin
fibrils in (a) are shown inside the yellow circles. The kymographs for anindividual selected fiber (shown inside pink rectangles in a and c) are
shown for amylin in the absence (b) and presence of PMAQA (d). The
kymographs show the growth of amylin fibers as a function of time(indicated with pink arrows) in the absence of PMAQA, whereas PMAQA
blocks amylin’s nucleation and seeding processes. The scale bar is 200
nm.

time using HS-AFM as the results presented above indicated anantagonistic behavior of PMAQA on amylin aggregation (Fig. 6). The
fibrillation kinetics of freshly prepared amylin monomers was
accelerated by adding preformed sonicated amylin amyloid seeds as
described in the Experimental Section. As shown in Figure 6a (toptrace), the addition of amylin monomers to pre-incubated amylin
amyloid seeds (at t=0 s) exhibited a steady growth of fibers (Movie
S1). Seeding reaction (or fibril growth) and de novo nucleation(binding of additional fibril seeds to the stage) were observed (Fig.
6a). At 1000 s, we observed substantial growth in fiber morphology,
de novo nucleated amylin fibers and several fibers were stopped
growing after encountering with other fibers at their growing end(Fig.6a, Movie S1). The growth of the selected amylin fiber (Fig. 6a,rectangle) in the absence of PMAQA are represented as kymographs
as a function of time in one direction. The kymograph showed asubstantial growth on the free end of the selected amylin fiber (Fig.

Fig. 7 TEM images of β-amyloid-1-40 and amylin interacting with
PMAQA. TEM images of 5 μM PMAQA (a) and 5 μM β-amyloid-1-40 in theabsence (b) or presence (c) of an equivalent concentration of PMAQA
incubated for 24 hours at room temperature under continuous agitation.
TEM images of sonicated β-amyloid-1-40 fibers (d) prepared frommatured fibers (see the Experimental section) in the presence of 5 μM
PMAQA (e); 5 μM β-amyloid-1-40 monomers (f); and 5 μM PMAQA and 5μM β-amyloid-1-40 monomers (g) incubated for 24 hours at room
temperature under continuous agitation. TEM images of 5 μM amylin in
the absence (h) or presence (i) of 5 μM PMAQA incubated for 24 hours atroom temperature under continuous agitation. TEM images of sonicatedamylin fibers (j) prepared from matured fibers (see the Experimental
section) in the presence of 5 μM PMAQA and 5 μM amylin monomers (k);and 5 μM PMAQA (l) incubated for 24 hours at room temperature undercontinuous agitation. The superscripts “M” and “F” denote the initial
species of the peptide as monomers and fibers, respectively, used for the
sample preparation. The inset in (l) shows the average width of amylin
fibers that were calculated from 10 distinct short amylin fibers selected
randomly using ImageJ plugin from (j) and (l).

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We further characterized the morphology of β-amyloid-1-40 andamylin species present under various sample conditions (see theExperimental section). TEM image of 5 µM PMAQA showed verysmall particles (Fig. 7a), whereas freshly dissolved β-amyloid-1-40
monomers (5 µM) after continuous agitation at room temperaturefor 24 hours showed fibril morphology (Fig. 7b). On the other hand,
β-amyloid-1-40 monomers (5 µM) incubated with an equimolar
concentration of PMAQA exhibited amorphous-like aggregates (Fig.
7c). This correlate with the observed ThT fluorescence results thatshowed a substantial reduction in fluorescence intensity (Fig. 1a). We
next examined the effect of PMAQA on β-amyloid-1-40 aggregationin the presence of amyloid seeds. TEM confirmed the formation ofshort fibrils when sonicating matured fibers for 2 hours (Fig. 7d). We
did not observe a significant change in the sonicated fiber
morphology incubated with equimolar PMAQA for 24 hours in theabsence of peptide monomers (Fig. 7e). However, a remarkabledifference in β-amyloid-1-40 morphology was observed when a selfseeding reaction was performed in the absence or presence of
PMAQA (Fig. 7f and g). The self-seeding reaction presented amyloid
fiber growth in the absence of PMAQA (Fig. 7f), whereas in thepresence of PMAQA short fibers and amorphous-like fiber
morphology were identified (Fig. 7g). Unlike β-amyloid-1-40, amylin
monomers (5 µM) incubated with equimolar PMAQA exhibited nofibers (Fig. 7i) which correlate to the ThT and CD results (Figs. 1b and2b). Moreover, sonicated amylin fibers incubated with 5 µM PMAQA
for 24 hours in the absence of monomers showed a relatively thickfibril morphology as compared to the short-sized sonicated fibers(Fig. 7j-l). Interestingly, sonicated amylin fibers in presence of
equimolar peptide monomers and PMAQA showed no fibril growthwhich correlates the HS-AFM observations (Fig. 7k). A quantitative
analysis of the widths of sonicated fibers (Fig. 7j) using ImageJ pluginpresented an average fiber width of ~10.79 nm. In the presence of
PMAQA, the sonicated fibers depicted an average width of ~15.69
nm indicating the possibility of PMAQA binding (Fig. 7l). Overall, theTEM analysis showed the formation of distinct morphological speciesof β-amyloid-1-40 and amylin in the presence or absence of PMAQA.
These results further highlighted the opposite behaviours of PMAQA
on the aggregation of β-amyloid-1-40 and amylin as observed inother biophysical experiments.

Conclusions

In conclusion, we have demonstrated the counter activities of acationic PMAQA polymer on two different amyloidogenic peptides
that are connected to AD and T2D. At substoichiometricconcentrations, PMAQA showed significant inhibitory activity inamylin aggregation; whereas it significantly accelerated b-amyloid-140’s aggregation by quickly altering the equilibrium state of bamyloid-1-40 from an unfolded structure to a β-sheet structure. Our
mechanistic study provides insights into the binding of PMAQA to bamyloid-1-40 or amylin at atomic-level that could be helpful inunderstanding the modulation of peptide self-assembly and could
aid in potential inhibitor designing. We believe that the opposite
aggregation kinetics of two different amyloidogenic proteins in thepresence of a cationic polymer delineated in this study are likely toopen avenues to test their potential therapeutic activities against anarray of amyloid proteins involved in other human amyloid diseases
by controlling functionalization of the polymer’s chemical property.

View Article Online

Conflicts of interest

DOI: 10.1039/C8SC05771K“There are no conflicts to declare”.

Acknowledgements

This study was supported by funds from NIH (AG048934 toA.R.). This work was (in part) performed under the International
Collaborative Research Program of Institute for Protein
Research, Osaka University, ICR-18-02.

References
1
2
3
4
5
6
7
8
9
10
11
12

13
14
15
16
17
18
19

20
21
22
23
24
25
26
27
28

T. P. J. Knowles, M. Vendruscolo and C. M. Dobson, Nat. Rev. Mol. Cell Biol.,2014, 15, 496–496.
S. A. Kotler, P. Walsh, J. R. Brender and A. Ramamoorthy, Chem. Soc. Rev.,
2014, 43, 6692–6700.
C. A. Ross and M. A. Poirier, Nat. Med., 2004, 10, S10–S17.
L. M. Young, A. E. Ashcroft and S. E. Radford, Curr. Opin. Chem. Biol., 2017,39, 90–99.
J. C. Sacchettini and J. W. Kelly, Nat. Rev. Drug Discov., 2002, 1, 267–275.
J. Luo, S. K. T. S. Wärmländer, A. Gräslund and J. P. Abrahams, J. Biol. Chem.,
2016, 291, jbc.R116.714576.
A. Abedini, A. Plesner, P. Cao, Z. Ridgway, J. Zhang, L. H. Tu, C. T. Middleton,
B. Chao, D. J. Sartori, F. Meng, H. Wang, A. G. Wong, M. T. Zanni, C. B.
Verchere, D. P. Raleigh and A. M. Schmidt, Elife, 2016, 5, e12977.
A. Spanopoulou, L. Heidrich, H.-R. Chen, C. Frost, D. Hrle, E. Malideli, K. Hille,
A. Grammatikopoulos, J. Bernhagen, M. Zacharias, G. Rammes and A.
Kapurniotu, Angew. Chemie Int. Ed., 2018, 57, 14503–14508.
Z. Liu, A. Zhang, H. Sun, Y. Han, L. Kong and X. Wang, RSC Adv., 2017, 7,
6046–6058.
S.-Y. Hung and W.-M. Fu, J. Biomed. Sci., 2017, 24, 47.
A. J. Doig, M. P. Del Castillo-Frias, O. Berthoumieu, B. Tarus, J. NasicaLabouze, F. Sterpone, P. H. Nguyen, N. M. Hooper, P. Faller and P.
Derreumaux, ACS Chem. Neurosci., 2017, 8, 1435–1437.
H. Kroth, A. Ansaloni, Y. Varisco, A. Jan, N. Sreenivasachary, N. RezaeiGhaleh, V. Giriens, S. Lohmann, M. P. López-Deber, O. Adolfsson, M.
Pihlgren, P. Paganetti, W. Froestl, L. Nagel-Steger, D. Willbold, T. Schrader,
M. Zweckstetter, A. Pfeifer, H. A. Lashuel and A. Muhs, J. Biol. Chem., 2012,
287, 34786–34800.
A. Pithadia, J. R. Brender, C. A. Fierke and A. Ramamoorthy, J. Diabetes Res.,
2016, 2016, 204632.
A. J. Doig and P. Derreumaux, Curr. Opin. Struct. Biol., 2015, 30, 50–56.
A. Granja, I. Frias, A. R. Neves, M. Pinheiro and S. Reis, Biomed Res. Int.,
2017, 2017, 5813793.
Q. Song, H. Song, J. Xu, J. Huang, M. Hu, X. Gu, J. Chen, G. Zheng, H. Chen
and X. Gao, Mol. Pharm., 2016, 13, 3976-3987.
M. Robinson, B. Yasie Lee and Z. Leonenko, AIMS Mol. Sci., 2015, 2, 332–
358.
M. Stefani and S. Rigacci, Int. J. Mol. Sci., 2013, 14, 12411–12457.
J. Bieschke, M. Herbst, T. Wiglenda, R. P. Friedrich, A. Boeddrich, F. Schiele,
D. Kleckers, J. M. Lopez Del Amo, B. A. Grüning, Q. Wang, M. R. Schmidt, R.
Lurz, R. Anwyl, S. Schnoegl, M. Fändrich, R. F. Frank, B. Reif, S. Günther, D.
M. Walsh and E. E. Wanker, Nat. Chem. Biol., 2012, 8, 93–101.
S. Derrick, R. A. Kerr, Y. Nam, S. B. Oh, H. J. Lee, K. G. Earnest, N. Suh, K. L.
Peck, M. Ozbil, K. J. Korshavn, A. Ramamoorthy, R. Prabhakar, E. J. Merino, J.
Shearer, J. Lee and B. T. Ruotolo, 2015, 137, 14785-14797.
M. Sato, K. Murakami, M. Uno, Y. Nakagawa, S. Katayama, K. I. Akagi, Y.
Masuda, K. Takegoshi and K. Irie, J. Biol. Chem., 2013, 288, 23212–23224.
J. Kang, S. J. C. Lee, J. S. Nam, H. J. Lee, M. G. Kang, K. J. Korshavn, H. T. Kim,
J. Cho, A. Ramamoorthy, H. W. Rhee, T. H. Kwon and M. H. Lim, Chem. - A
Eur. J., 2017, 23, 1645–1653.
B. Sahoo, T. Genjo, M. Bekier II, S. N. Cox, A. Stoddard, M. Ivanova, K.
Yasuhara, C. Fierke, Y. Wang and A. Ramamoorthy, Chem. Commun., 54,
12883-12886.
Y. Song, E. G. Moore, Y. Guo and J. S. Moore, J. Am. Chem. Soc., 2017, 139,
4298–4301.
E. H. Pilkington, M. Lai, X. Ge, W. J. Stanley, B. Wang, M. Wang, A. Kakinen,
M.-A. Sani, M. R. Whittaker, E. N. Gurzov, F. Ding, J. F. Quinn, T. P. Davis and
P. C. Ke, Biomacromolecules, 2017, 18, 4249-4260.
C. Cabaleiro-Lago, O. Szczepankiewicz and S. Linse, Langmuir, 2012, 28,
1852–1857.
B. R. Sahoo, T. Genjo, S. J. Cox, A. K. Stoddard, G. M. Anantharamaiah, C.
Fierke and A. Ramamoorthy, J. Mol. Biol., 2018, 430, 4230-4244.
J. Luo, C. H. Yu, H. Yu, R. Borstnar, S. C. L. Kamerlin, A. Gräslund, J. P.
Abrahams and S. K. T. S. Wärmländer, ACS Chem. Neurosci., 2013, 4, 454–

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the inhibitory activity of PMAQA on amylin aggregation at amolecular-level, which is consistent with other experimental results
reported in this study.

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29
30
31
32

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33
34
35
36
37
38
39
40
41
42
43

44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69

ARTICLE

462.
A. Assarsson, S. Linse and C. Cabaleiro-Lago, Langmuir, 2014, 30, 8812–
8818.
Y. Li, M. Cao and Y. Wang, J. Phys. Chem. B, 2006, 110, 18040–18045.
K. Yasuhara, J. Arakida, T. Ravula, S. K. Ramadugu, B. Sahoo, J.-I. Kikuchi and
A. Ramamoorthy, J. Am. Chem. Soc., 2017, 139, 18657-18663.
W. Wang, A. Shao, N. Zhang, J. Fang, J. J. Ruan and B. H. Ruan, Sci. Rep.,
2017, 7, 1–10.
D. Paolino, A. Vero, D. Cosco, T. M. G. Pecora, S. Cianciolo, M. Fresta and R.
Pignatello, Front. Pharmacol., 2016, 7, 1–9.
K. Garai, S. L. Crick, S. M. Mustafi and C. Frieden, Protein Expr. Purif., 2009,
66, 107–112.
M. Dasari, A. Espargaro, R. Sabate, J. M. Lopez Del Amo, U. Fink, G. Grelle, J.
Bieschke, S. Ventura and B. Reif, ChemBioChem, 2011, 12, 407–423.
P. Schanda and B. Brutscher, J. Am. Chem. Soc., 2005, 127, 8014–8015.
TD. Goddard and D. G. Kneller, Univ. California, San Fr., 2004, 14, 15.
T. Ando, N. Kodera, E. Takai, D. Maruyama, K. Saito and A. Toda, Proc Natl
Acad Sci U S A, 2001, 98, 12468–12472.
T. Uchihashi, N. Kodera and T. Ando, Nat. Protoc., 2012, 7, 1193–1206.
T. Watanabe-Nakayama and K. Ono, High-speed atomic force microscopy ofindividual amyloidogenic protein assemblies, 2018, 1814, 201-212.
A. K. Malde, L. Zuo, M. Breeze, M. Stroet, D. Poger, P. C. Nair, C. Oostenbrink
and A. E. Mark, J. Chem. Theory Comput., 2011, 7, 4026–4037.
S. Vivekanandan, J. R. Brender, S. Y. Lee and A. Ramamoorthy, Biochem.
Biophys. Res. Commun., 2011, 411, 312–316.
D. C. Rodriguez Camargo, K. Tripsianes, K. Buday, A. Franko, C. Göbl, C.
Hartlmüller, R. Sarkar, M. Aichler, G. Mettenleiter, M. Schulz, A. Böddrich, C.
Erck, H. Martens, A. K. Walch, T. Madl, E. E. Wanker, M. Conrad, M. H. De
Angelis and B. Reif, Sci. Rep., 2017, 7, 44041.
D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A. E. Mark and H. J. C.
Berendsen, J. Comput. Chem., 2005, 26, 1701–1718.
N. Schmid, A. P. Eichenberger, A. Choutko, S. Riniker, M. Winger, A. E. Markand W. F. Van Gunsteren, Eur. Biophys. J., 2011, 40, 843–856.
W. Humphrey, A. Dalke and K. Schulten, J. Mol. Graph., 1996, 14, 33–38.
P. A. Kollman, I. Massova, C. Reyes, B. Kuhn, S. Huo, L. Chong, M. Lee, T. Lee,
Y. Duan, W. Wang, O. Donini, P. Cieplak, J. Srinivasan, D. A. Case and T. E.
Cheatham, Acc. Chem. Res., 2000, 33, 889-897.
D. Spiliotopoulos, A. Spitaleri and G. Musco, PLoS One, 2012, 7, e46902.
B. R. Sahoo, J. Maharana, M. C. Patra, G. K. Bhoi, S. K. Lenka, P. K. Dubey, S.
Goyal, B. Dehury and S. K. Pradhan, Colloids Surfaces B Biointerfaces, 2014,
121, 307–318.
B. R. Sahoo, J. Maharana, G. K. Bhoi, S. K. Lenka, M. C. Patra, M. R. Dikhit, P.
K. Dubey, S. K. Pradhan and B. K. Behera, Mol. BioSyst., 2014, 10, 1104–
1116.
Y. Yoshimura, Y. Lin, H. Yagi, Y.-H. Lee, H. Kitayama, K. Sakurai, M. So, H. Ogi,
H. Naiki and Y. Goto, Proc. Natl. Acad. Sci., 2012, 109, 14446–14451.
A. Micsonai, F. Wien, L. Kernya, Y.-H. Lee, Y. Goto, M. Réfrégiers and J.
Kardos, Proc. Natl. Acad. Sci., 2015, 112, E3095–E3103.
Q. Wang, N. Shah, J. Zhao, C. Wang, C. Zhao, L. Liu, L. Li, F. Zhou and J.
Zheng, Phys. Chem. Chem. Phys., 2011, 13, 15200.
K. Ono, M. M. Condron and D. B. Teplow, Proc. Natl. Acad. Sci., 2009, 106,14745–14750.
A. B. Soriaga, S. Sangwan, R. MacDonald, M. R. Sawaya and D. Eisenberg, J.
Phys. Chem. B, 2016, 120, 5810–5816.
V. Castelletto, I. W. Hamley, P. J. Harris, U. Olsson and N. Spencer, J Phys
Chem B, 2009, 113, 9978–9987.
T. Guterman, M. Kornreich, A. Stern, L. Adler-Abramovich, D. Porath, R.
Beck, L. J. W. Shimon and E. Gazit, Nat. Commun., 2016, 7, 1–10.
T. J. Esparza, N. C. Wildburger, H. Jiang, M. Gangolli, N. J. Cairns, R. J.
Bateman and D. L. Brody, Sci. Rep., 2016, 6, 1–16.
Y. Bram, A. Frydman-Marom, I. Yanai, S. Gilead, R. Shaltiel-Karyo, N.
Amdursky and E. Gazit, Sci. Rep., 2014, 4, 1–9.
A. Assarsson, E. Hellstrand, C. Cabaleiro-Lago and S. Linse, ACS Chem.
Neurosci., 2014, 5, 266–274.
P. J. Marek, V. Patsalo, D. F. Green and D. P. Raleigh, Biochemistry, 2012, 51,
8478–8490.
J. Miguel, U. Fink, M. Dasari, G. Grelle, E. E. Wanker, J. Bieschke and B. Reif,
J. Mol. Biol., 2012, 421, 517–524.
A. Gladytz, B. Abel and H. J. Risselada, Angew. Chemie - Int. Ed., 2016, 55,
11242–11246.
M. Wang, A. Kakinen, E. H. Pilkington, T. P. Davis and P. C. Ke, Biomater. Sci.,
2017, 5, 485–493.
B. K. Yoo, Y. Xiao, D. McElheny and Y. Ishii, J. Am. Chem. Soc., 2018, 140,
2781–2784.
Y. Miller, B. Ma and R. Nussinov, Proc. Natl. Acad. Sci., 2010, 107, 9490–
9495.
V. S. Mithu, B. Sarkar, D. Bhowmik, M. Chandrakesan, S. Maiti and P. K.
Madhu, Biophys. J., 2011, 101, 2825–2832.
S. Chakraborty and P. Das, Sci. Rep., 2017, 7, 1–12.
M. Guo, P. M. Gorman, M. Rico, A. Chakrabartty and D. V. Laurents, FEBS

70
71
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Lett., 2005, 579, 3574–3578.
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M. A. Grant, N. D. Lazo, A. Lomakin, M. M. Condron, H. Arai, G. Yamin, A. C.
DOI: 10.1039/C8SC05771K
Rigby and D. B. Teplow, Proc. Natl. Acad. Sci., 2007, 104, 16522–16527.
T. Watanabe-Nakayama, K. Ono, M. Itami, R. Takahashi, D. B. Teplow and M.
Yamada, Proc. Natl. Acad. Sci., 2016, 113, 5835–5840.
P. E. Milhiet, D. Yamamoto, O. Berthoumieu, P. Dosset, C. le Grimellec, J. M.
Verdier, S. Marchal and T. Ando, PLoS One, 2010, 5, e13240.

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