Self-assembly of a model peptide incorporating a hexa-histidine sequence attached to an Oligo-Alanine sequence, and binding to gold NTA/nickel nanoparticles

Article abstract of DOI:10.1021/bm500950c

Amyloid fibrils are formed by a model surfactant-like peptide (Ala)₁₀-(His)₆ containing a hexa-histidine tag. This peptide undergoes a remarkable two-step self-assembly process with two distinct critical aggregation concentrations (cac's), probed by fluorescence techniques. A micromolar range cac is ascribed to the formation of prefibrillar structures, whereas a millimolar range cac is associated with the formation of well-defined but more compact fibrils. We examine the labeling of these model tagged amyloid fibrils using Ni-NTA functionalized gold nanoparticles (Nanogold). Successful labeling is demonstrated via electron microscopy imaging. The specificity of tagging does not disrupt the β-sheet structure of the peptide fibrils. Binding of fibrils and Nanogold is found to influence the circular dichroism associated with the gold nanoparticle plasmon absorption band. These results highlight a new approach to the fabrication of functionalized amyloid fibrils and the creation of peptide/nanoparticle hybrid materials.

Full text of DOI:10.1021/bm500950c

Article
pubs.acs.org/Biomac
Self-Assembly of a Model Peptide Incorporating a Hexa-Histidine
Sequence Attached to an Oligo-Alanine Sequence, and Binding to
Gold NTA/Nickel Nanoparticles
Ian W. Hamley,* Steven Kirkham, Ashkan Dehsorkhi, and Valeria Castelletto†
Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom
Jozef Adamcik and Raffaele Mezzenga
Food and Soft Materials Science, ETH Zurich, Schmelzbergstrasse 9, 8092 Zurich, Switzerland
̈
̈
Janne Ruokolainen
Department of Applied Physics, Aalto University School of Science, P.O. Box 15100, FI-00076 Aalto, Finland
Claudia Mazzuca, Emanuela Gatto, and Mariano Venanzi
Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Via Ricerca Scientifica 1, 00133 Rome, Italy
Ernesto Placidi
Institute of Structure of Matter, CNR, Department of Physics, University of Rome “Tor Vergata”, 00133 Rome, Italy
Panayiotis Bilalis and Hermis Iatrou
University of Athens, Department of Chemistry, Panepistimiopolis Zografou, 157 71 Athens, Greece
S
* Supporting Information
ABSTRACT: Amyloid fibrils are formed by a model
surfactant-like peptide (Ala)10-(His)6 containing a hexa-
histidine tag. This peptide undergoes a remarkable two-step
self-assembly process with two distinct critical aggregation
concentrations (cac’s), probed by fluorescence techniques. A
micromolar range cac is ascribed to the formation of
prefibrillar structures, whereas a millimolar range cac is
associated with the formation of well-defined but more
compact fibrils. We examine the labeling of these model
tagged amyloid fibrils using Ni-NTA functionalized gold
nanoparticles (Nanogold). Successful labeling is demonstrated
via electron microscopy imaging. The specificity of tagging
does not disrupt the β-sheet structure of the peptide fibrils. Binding of fibrils and Nanogold is found to influence the circular
dichroism associated with the gold nanoparticle plasmon absorption band. These results highlight a new approach to the
fabrication of functionalized amyloid fibrils and the creation of peptide/nanoparticle hybrid materials.
widely used. Since nickel ions are hexa-coordinated and four
electrons are used in binding to NTA, two electrons are
available to bind hexahistidine tags. This interaction has high
INTRODUCTION
■
The hexa-histidine tag, developed by researchers at Roche,1 is
widely used in the affinity purification of expressed recombinant
proteins.2 The protein to be labeled is engineered to
incorporate N- or C-terminal histidine repeats. A common
system employs a hexahistidine tag along with a metal chelate
among which the nitrilotriacetic acid (NTA)/Ni2+ system is
Received: July 1, 2014
Revised: August 4, 2014
Published: August 8, 2014
© 2014 American Chemical Society
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Scheme 1. Concept of Nanogold-Decorated Amyloid Fibrils with Hexahistidine Exterior
affinity and selectivity.3−5 NTA/nickel functionalized nano-
particles termed Nanogold are available commercially for the
decoration of hexahistidine-tagged proteins for transmission
electron microscopy (TEM) imaging,6,7 and this has been used
to image a variety of protein complexes. The interaction of
hexahistidine-tagged proteins (derived from the cartilage
oligomeric matrix protein (C) coiled-coil domain, His6-C)
with divalent metal ions has been investigated.8 Binding to Zn2+
confers enhanced helical structure and stability, while Ni2+
promotes aggregation. To our knowledge, this high affinity
labeling technique has not been employed to image protein or
peptide “amyloid” fibrils. Amyloid refers to the formation of β-
sheet rich fibrillar aggregates by peptides or proteins which
results from misfolding induced by partial denaturation.
Prior work shows that organic nanoparticles (NPs) can
influence amyloid fibrillization, potentially either inhibiting or
accelerating fibrillization, dependent on the nature of the
interaction of the peptide with the nanoparticle surface.9
Inorganic nanoparticles such as CdSe/ZnS quantum dots have
been used to label amyloid-forming peptides to probe the fibril
formation or disruption process.10−12 The interaction of gold
NPs with amyloid fibrils has been examined, and in general
inclusion of these nanoparticles appears to disfavor amyloid
formation. In many cases, however, the NPs have a specific
coating, which may be useful in applications including immune
adjuvants (since the NPs can stimulate a macrophage
response)13 or in disrupting fibrillization14−17 which may
reduce amyloid cytotoxicity for ultimate therapeutic applica-
tions.9 Recently, amyloid−metal nanoparticle conjugates for
controlled transport in living cells and therapeutics have been
proposed.18 Localized heating using microwave irradiation of
gold NPs can also induce amyloid fibril disassembly.19,20 In
some cases, gold NPs can induce aggregation, an effect ascribed
to the presence of misfolded proteins at the nanoparticle
surface.21 In all of these prior studies, however, specific binding
interactions between the amyloid peptide and the gold NPs
were not utilized. Very recently, curli amyloid fibrils expressed
by E. coli with a histidine tag have been tagged with Ni-NTA
Nanogold.22 Matsui and co-workers investigated the use of
histidine-rich peptide to produce gold nanowires.23 The
histidine-rich peptide AHHAHHAAD was deposited onto
nanowires formed by the amphiphile heptane dicarboxylate.
The immobilization of the peptide on the nanowires facilitates
subsequent coating with gold via reduction of a gold salt.
In the present paper, we investigate the possibility to
decorate model “amyloid” fibrils containing the high affinity
hexa-histidine tag with Ni-NTA gold nanoparticles termed
“Nanogold”. The first part of the Article describes the self-
assembly properties of the peptide (Ala)10-(His)6, A10H6, and
this is then followed by our results which show that the
nanoparticles do not disrupt fibrillization, and thus that it is
possible to successfully decorate amyloid-like fibrils with the
gold nanoparticles. It has very recently been shown that gold
nanoparticles coated with thiol or citrate functionalities can be
coated with histidine-tagged proteins,24 and Nanogold has been
used to tag histidine-tagged proteins, for enhanced TEM
imaging.7 In contrast to this work, our research involves the
specific histidine/Ni-NTA binding motif and employs model
amyloid-forming peptides. In contrast to the work of Matsui et
al.,23 we did not require additional amphiphiles as templates to
create peptide nanofibrils, since these are formed directly by
A10H6.
EXPERIMENTAL SECTION
■
Synthesis of Alanine10-Histidine6 (A10H6) Oligopeptides. Boc-
His(Trt)-OH was obtained from Christof Senn Laboratories (>99%),
and alanine from Aldrich (97%). Dimethylamine (DMA, >99.9%,
Aldrich, bp 7 °C) was condensed under high vacuum at −78 °C and
was treated with sodium hydroxide pellets at room temperature for 1
day, and was subsequently distilled into precalibrated ampules with
break-seals. It was then diluted with N,N′-dimethylformamide (DMF)
to the appropriate concentration in a sealed apparatus equipped with
precalibrated ampules and kept away from light. Thionyl chloride
(99.7%, Acros Organics) was distilled prior use. Triphosgene (99%)
was purchased from Acros Organics and used as received. Triethyl-
amine (>99%, Acros Organics) was dried over calcium hydride for 1
day and then distilled and stored in the vacuum line over sodium.
Ethyl acetate (>99.5%, Merck) was fractionally distilled over
phosphorus pentoxide. Hexane (>99%, Merck) was distilled over
sodium. Purification of tetrahydrofuran (THF, dried, max 0.005%
water, Merck) was performed using standard high vacuum techniques
reported elsewhere.25 DMF (Fisher 99.9+ %, special grade for peptide
synthesis with less than 50 ppm of active impurities) was further
purified by short-path fractional distillation under high vacuum. The
middle fraction was always used.
Synthesis of Nim Trityl Protected N-Carboxyanhydride of L-
Histidine (Trt-His-NCA). The synthesis of Trt-His-NCA was
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performed in two steps as previously reported.26 In the first step, the
HCl salt of Trt-His-NCA was synthesized, followed by removal of the
HCl to produce the pure Trt-His-NCA monomer. Briefly, 20 g (40.2
mmol) of Boc-His(Trt)-OH was added and dried overnight under
high vacuum. Then 150 mL of THF was distilled in the flask, giving a
clear yellowish solution. The reaction flask was placed in an ice-bath,
filled with argon, and 3.25 mL (44.2 mmol) of thionyl chloride diluted
in 20 mL of THF was added dropwise in a period of 10 min. By the
end of addition of thionyl chloride, the solution became yellowish.
After 2 h, the solution was poured into 2 L of cold (Et)2O with
precipitation of Trt-His-NCA·HCl as the major product. The solid was
filtered and then transferred to a round-bottom flask. Next, 300 mL of
ethyl acetate was distilled and the mixture was placed into a water bath,
at 45 °C for 1 h, resulting in dissolution. Then the solution was cooled
to 0 °C with an ice bath and Trt-His-NCA·HCl was formed as a
precipitate which was isolated as the only product after filtration (12.5
g, 28 mmol).
In the second step, 200 mL of ethyl acetate was distilled to the
above solid and the suspension was placed in an ice bath. A solution of
50 mL ethyl acetate with an equimolar amount of triethylamine (3.5
mL, 28 mmol) was slowly added dropwise under vigorous stirring
(duration of addition 1 h). The resulting triethylamine hydrochloride
was filtered off, and the filtrate was poured into 1.5 L of nonsolvent
hexane in order to recrystallize the Trt-His-NCA. A second
recrystallization occurred with a mixture of solvent/nonsolvent ethyl
acetate/hexane (1:5), and the white solid was isolated by filtration.
Finally, Trt-His-NCA was dried under vacuum overnight (11.05 g, 27
mmol, 67% yield).
Synthesis of N-Carboxy Anhydride of L-Alanine (Ala-NCA).
The synthesis process of the Alanine NCA (Ala-NCA) was
accomplished according to a previously published method.27 Briefly,
Ala-NCA was synthesized from the corresponding L-α-amino acid and
triphosgene in acetonitrile (suspension) at 70 °C, under an inert
atmosphere. The unreacted species along with the amino acid salts
(insoluble species) were removed by filtration. Ala-NCA was
subsequently dissolved and dried several times with ethyl acetate
under high vacuum, in order to remove the excess triphosgene, which
sublimes under high vacuum, along with the remaining HCl. Finally,
Ala-NCA was dissolved in ethyl acetate and was recrystallized from n-
hexane three times under high vacuum in a custom-made apparatus.
The purified NCA was stored under an inert atmosphere at 0 °C.
Synthesis of A10H6 Oligopeptide. Polymerization was carried
out with dimethylamine as the initiator. The sequential addition
methodology was used for the synthesis of the oligopeptide. In a
solution of Ala-NCA in DMF (6 mL, ∼10% w/w), 5.2 × 10−4 mol
dimethylamine was added. After the completion of polymerization (2
days), Trt-His-NCA (1.31 g, 3.1 × 10−3 mol) was then added and was
left to react until all monomer was completely consumed. After
complete consumption of Trt-His-NCA (4 days), a small aliquot of the
raw product was removed from the reactor for characterization and
control of the completion of the polymerization. The final
oligopeptide was precipitated in diethyl ether, filtered, and dried to
constant weight.
at a rate of 1 mL/min at 60 °C. The SEC chromatograms obtained for
the A10 precursor along with the final A10H6 are shown in Supporting
Information Figure 1. The molecular weight of the A10 precursor
obtained is 780 g/mol which corresponds to the 10 alanine units plus
the molecular weight of the initiator. The molecular weight of the final
block oligopeptide was 3050 g/mol. Consequently, the molecular
weight of the protected oligohistidine block was 2270 which
corresponds to 6 protected histidine units. The corresponding
molecular weight of the deprotected oligohistidine in the form of
TFA salt would be 1500. The deprotection of trityl groups of His
segments was confirmed by the elimination of the peaks at 708 and
728 cm−1 in the FTIR spectra (Supporting Information Figure 2).
The composition of the oligopeptide was verified by 1H NMR
1
spectroscopy (Supporting Information Figure 3). H NMR spectros-
copy (300 MHz) was performed using a Varian Unity Plus 300/54
spectrometer. The spectra of the polymers were acquired in
CF3COOD at room temperature. The peaks used to obtain the
composition were at 4.85 ppm which is the total α-protons of
oligoalanine segments and the one at 5.25 ppm which is the total α-
protons of the oligohistidine groups. The molar ratio between A/H
groups was found to be 10/6.
Other Reagents. MOPS buffer was purchased from Sigma-Aldrich,
and 5 nm Ni-NTA-Nanogold was purchased from Nanoprobes.
MOPS was prepared by dissolving powder in distilled water to give a
concentration of 50 mM. A10H6 was dissolved through vortex and bath
sonication in distilled water for initial experiments, and then 50 mM
MOPS at pH 7.9 was used for experiments incorporating Nanogold
particles. The Nanogold particle preparation protocol indicated that
His-tagged protein samples were to be preprepared in binding buffer
(MOPS in this case) at pH 7−8, which was adjusted using 8.4%
NaOH. This was to avoid protonation of the histidines which would
affect interactions with the metal. The A10H6 samples were incubated
with the Nanogold particles in MOPS buffer for 5−30 min at room
temperature.
Steady State Fluorescence Experiments. Fluorescence spec-
troscopy was used to locate the critical aggregation concentration in
the micromolar (cac1) and millimolar (cac2) concentration regions.
Fluorescence spectra were recorded with a Varian Cary Eclipse
fluorescence spectrometer with samples in 4 mm inner width quartz
cuvettes. Thioflavin T (ThT) was used as a probe of amyloid fibril
formation, and pyrene as a fluorophore sensitive to the hydrophobic
environment. The fluorescence assays were performed using 4.9 ×
10−3−1.95 wt % A10H6 solutions in distilled water. The concentration
of ThT used was 5.5 × 10−9 wt %, and that of pyrene was 1.3 × 10−5
wt %. For the ThT assay, spectra were measured from 460 to 600 nm
using λex = 440 nm. For the pyrene assays, spectra were measured from
360 to 550 nm using λex = 339 and 1.5 nm slit width for both
excitation and emission monochromators.
To detect cac1, the ratio of the intensities of the first (I1, λem = 373
nm) and third (I3, λem = 382 nm) vibrational components of the
lowest energy (S2−S0) fluorescence emission of pyrene was monitored
as a function of increasing peptide concentration in the buffer
solution.28 Measurements were carried out at 25 °C using a Fluorolog
For the deprotection of trityl groups, the oligopeptide was
suspended in CH2Cl2 (20% w/v) and an equal volume of
trifluoroacetic acid (TFA) was added. The polymer was completely
dissolved and was left to be deprotected for 1 h at room temperature,
and the solution became yellowish-light brown. Subsequently, an
equimolar amount of triethyl silane was added (in respect to the
number of His monomeric units), and the solution from yellowish-
light brown turned colorless. The solution was poured in diethyl ether,
and the white solid was filtered and dried. The reactions used are
shown in Supporting Information Scheme 1.
III spectrofluorimeter (HORIBA, Japan). Samples were excited at λex =
310 nm, and emission was recorded in the 350−450 nm spectral range.
Experiments were performed at two different pH values, that is, at pH
= 7.6 and pH = 4.4. In the first case, a Tris buffer ([Tris] = 25 mM,
[NaCl] = 150 mM) was used, while in the second case a phosphate
buffer ([NaH2PO4] = 25 mM, [NaCl] = 150 mM) was used. The dye
concentration was 4 × 10−5 wt % (2 μM), while the peptide
concentration varied from 0 to 0.007 wt % (100 μM). Peptide was
added from a concentrated methanol stock solution; the final dilution
was below 5%.
Characterization of Synthesized Oligopeptide. A special set of
columns for low molecular weight samples were used to characterize
the oligomers. The size exclusion chromatography (SEC) system
comprised of a Waters 600 high pressure liquid chromatographic
pump, Waters HT columns, a Waters 410 differential refractometer
detector, and a Precision PD 2020 two angles (15°, 90°) light
scattering detector. A 0.1 N LiBr DMF solution was used as an eluent
Time Resolved Fluorescence Experiments. To determine the
aggregation number at micromolar concentration, the fluorescence
decay of pyrene ([pyrene] = 2 μM; excitation: λex = 344 nm, emission:
λem = 400 nm) was progressively quenched by the water-insoluble
quencher, N,N′-dibutyl aniline (DBA).29 The quencher was
introduced in microliter aliquots of a concentrated ethanol solution
in the concentration range 0−50 μM. To investigate the dependence
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of the aggregation number on the peptide concentration, measure-
ments were performed with [A10H6] = 226 μM and [A10H6] = 400
μM. Experiments were performed in Tris buffer ([Tris] = 25 mM,
[NaCl] = 150 mM, pH = 7.6). Fluorescence decays were measured
using a Lifespec ps, SPC lifetime apparatus (Edinburgh Instruments,
U.K.). Excitation was achieved by using a diode (λex = 344 nm, pulse
duration <1 ns) at λem = 400 nm. The experimental time decays were
fitted through an iterative reconvolution procedure assuming that the
fluorescence intensity time decay of the probe (pyrene) in the
presence of the quencher (DBA) was accounted for by the equation:30
radial averaging) was performed using dedicated beamline software
ISPYB.
Cryo-Transmission Electron Microscopy (cryo-TEM). Experi-
ments were carried out using a field emission cryo-electron microscope
(JEOL JEM-3200FSC) operating at 200 kV. Images were taken using
bright-field mode and zero loss energy filtering (omega type) with a
slit with 20 eV. Micrographs were recorded using a Gatan Ultrascan
4000 CCD camera. The specimen temperature was maintained at
−187 °C during the imaging. Vitrified specimens were prepared using
an automated FEI Vitrobot device using Quantifoil 3.5/1 holey carbon
copper grids with 3.5 μm hole sizes. Grids were cleaned using a Gatan
Solarus 9500 plasma cleaner just prior to use and then transferred into
an environmental chamber of the FEI Vitrobot at room temperature
and 100% humidity. Thereafter, 3 μL of sample solution at 2 wt %
concentration was applied on the grid, blotted once for 1 s, and then
vitrified in a 1/1 mixture of liquid ethane and propane at −180 °C.
Grids with vitrified sample solutions were maintained in a liquid
nitrogen atmosphere and then cryo-transferred into the microscope.
Transmission Electron Microscopy (TEM). TEM imaging was
performed using a Philips CM20 transmission electron microscope
operated at 200 kV. Droplets of solutions were placed on Cu grids
coated with a carbon film (Agar Scientific, U.K.), stained with 1 wt %
uranyl acetate, and air-dried.
X-ray Diffraction (XRD). X-ray diffraction was performed on a
peptide stalk prepared by drawing a fiber of a 6% peptide solution
between the ends of wax-coated capillaries, after separation and drying
a stalk was left on the end of one capillary. The capillary was mounted
vertically onto the four axis goniometer of a RAXIS IV++ X-ray
diffractometer (Rigaku) equipped with a rotating anode generator.
The XRD data was collected using a Saturn 992 CCD camera. The
sample-to-detector distance was 50 mm.
Dynamic Light Scattering (DLS). Experiments were performed
using an ALV CGS-3 system with 5003 multidigital correlator. The
light source was a 20 mW He−Ne laser, linearly polarized, with λ =
633 nm. The scattering angle θ = 90° was used for all the experiments.
Samples were loaded into standard 0.5 cm diameter cylindrical glass
cells. DLS experiments measured the intensity correlation function of
the radiated light g(2)(q,t):32
I(t) = I(0)(exp{−t/τ0 − C[1 − exp(−kqt)]}
(1)
where τ0 is the time decay of the probe (measured separately under
the same experimental conditions, but in the absence of quencher
molecules), kq is the quenching rate constant, and C is the average
number of quenchers per aggregate. This equation assumes that the
fluorescence lifetime of the probe is much shorter than its residence
time in the hydrophobic core of the peptide aggregate. The
aggregation number (N) is then obtained using the equation:31
N = ([P] − cac1)C/[DBA]
(2)
where [P] is the peptide molar concentration, [DBA] is the molar
concentration of the quencher, and cac1 is the critical concentration
for the formation of prefibrillar aggregates.
Gold Nanoparticle UV/Vis Absorption Spectroscopy. Absorp-
tion measurements were carried out using a Cary 100 SCAN (Varian,
Palo Alto, CA) spectrophotometer. The Ni-NTA gold nanoparticle
solution (0.5 μM) was diluted 1:5 (v/v) with MOPS buffer (50 mM).
The experiment was carried out in a quartz cell of 1.0 cm path length.
Circular Dichroism (CD). For secondary structure measurements,
spectra for samples solutions in MOPS buffer made up in D2O (the
same samples being used for FTIR experiments) were recorded using a
Chirascan spectropolarimeter (Applied Photophysics, U.K.). The
sample was placed in a coverslip cuvette (0.1 mm thick). Spectra are
presented with absorbance A < 2 at any measured point with a 0.5 nm
step, 1 nm bandwidth, and 1 s collection time per step at 20 °C. The
post-acquisition smoothing tool from Chirascan software was used to
remove random noise elements from the averaged spectra. A residual
plot was generated for each curve in order to verify whether the
spectrum has been distorted during the smoothing process. The CD
signal from MOPS buffer was subtracted from the CD data of the
peptide solutions.
For dichroism studies of the histidine-gold nanoparticle interaction
in the visible region, CD experiments were performed using a Jasco
J600 spectrometer (Jasco, Tokyo, Japan) using quartz cuvettes of 1 cm
path length. For each spectrum, eight scans were recorded. Aliquots of
A10H6 solutions (72 μM, i.e., above cac1, and 7 μM, i.e., below cac1)
were added to 0.1 μM Ni-NTA gold nanoparticles in MOPS at pH 7.9.
Fourier Transform Infrared (FTIR) Spectroscopy. Spectra were
recorded for samples dissolved in MOPS buffer made up in D2O using
a Thermo Scientific Nicolet IS5 or a Nexus-FTIR spectrometer, both
equipped with a DTGS detector. Samples of around 10−20 μL were
placed between two CaF2 plates with a 0.0012 mm thick spacer in
between, and loaded into an Omnicell holder (Specac). Spectra were
scanned 128 times over the range of 900−4000 cm−1.
Small-Angle X-ray Scattering (SAXS). Experiments were
performed on beamline BM29 at the ESRF (Grenoble, France). A
few microlitres of samples were injected via an automated sample
exchanger at a slow and very reproducible flux into a quartz capillary
(1.8 mm internal diameter), which was then placed in front of the X-
ray beam. The quartz capillary was enclosed in a vacuum chamber, in
order to avoid parasitic scattering. After the sample was injected in the
capillary and reached the X-ray beam, the flow was stopped during the
SAXS data acquisition. The sample was thermostated throughout its
entire travel from the injector to the quartz capillary. SAXS
experiments were performed at 20 °C. The q = 4π sin θ/λ range is
approximately 0.04−5 nm−1, with λ = 1.03 Å (12 keV) and a 2.87 m
sample−detector distance. The images were captured using a
PILATUS 1 M detector. Data processing (background subtraction,
g2(q, t) = 1 + A[g(1)(q, t)]2
(3)
where A is an instrumental correction factor, q = [4πn sin(θ/2)]/λ is
the scattering vector (λ = vacuum wavelength of the radiation and n =
refractive index of the medium), t is the delay time, and g(1)(q,t) is the
electric field correlation function. The program CONTIN can be used
to determine the relaxation rate distribution of the system33 by
modeling of the field correlation function according to
g(1)(t) = ∞ G(Γ) exp(−Γt) dΓ
∫
(4)
0
where G(Γ) is the relaxation rate distribution. CONTIN allows for the
inverse Laplace transform in eq 4 and provides a tool for calculating
the size distribution of the system (G(RH); RH = hydrodynamic
radius). RH is taken as the radius corresponding to the maximum in
G(RH).
Atomic Force Microscopy (AFM). A 20 μL of solution (0.5 wt %
solution of A10H6 with Nanogold in MOPS) was deposited onto
freshly cleaved mica, incubated for 2 min, rinsed with Milli-Q water (ρ
= 18 MΩ cm−1), and dried by air. Tapping mode AFM was carried out
on a Nanoscope 8 multimode scanning force microscope (Bruker).
AFM cantilevers (Bruker) for use in soft tapping conditions were used
at a vibrating frequency of 150 kHz. Images were simply flattened
using the Nanoscope 8.1 software, and no further image processing
was carried out.
Additional AFM measurements on peptide films dried on a mica
surface were performed in air using a Veeco Multiprobe IIIa (Santa
Barbara, CA) instrument. Experiments were carried out at room
temperature (20 °C) in the tapping mode by using NanoSensors SiO2
tips with a force constant of about 40 N m−1 and a typical tip curvature
radius of 7 nm. An aliquot of 35 μL of the peptide sample (357 μM)
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was deposited onto a freshly cleaved mica surface and left to dry
completely in air before imaging. In the case of the mixed solution
peptide (250 μM) and Nanogold (0.02 μM), 10 μL of the solution was
deposited onto a freshly cleaved mica surface and left to dry
completely in air before imaging.
RESULTS
■
We assessed the presence of critical aggregation concentrations
using fluorescence assays with both pyrene and Thioflavin T.
Pyrene is a fluorophore sensitive to the local hydrophobic
environment,28,34 and has been used previously to determine
the cac for peptide systems.35−38 Thioflavin T is used as an
amyloid-specific dye.36,37 Assays using two types of dye were
performed, which revealed two separate aggregation processes.
We found that a critical aggregation concentration exists in the
micromolar concentration (cac1) range above which the
peptide A10H6 self-assembles. The concentration-dependence
of is the I1/I3 fluorescence intensity ratio of pyrene shown in
Figure 1.
Figure 2. Assays to determine the critical aggregation concentration
(cac2) of A10H6. (a) Pyrene fluorescence I1 (measured at 373 nm) and
(b) thioflavin T fluorescence (measured at 485 nm).
residues into the aggregate core. As described by eq 2, it was
possible to determine the association number of the micelle-like
aggregates from time-resolved pyrene fluorescence quenching
as described in the Experimental Section. A representative
fluorescence decay curve is shown in Supporting Information
Figure 6. An aggregation number N = (30 3) was determined
for a 150 μM solution, whereas N = (81 4) was obtained
from a 400 μM solution. The concentration-dependent values
of N show that the formation of prefibrillar aggregates is a
partially open equilibrium process, since N continues to
increase above cac1 (but well below cac2 for fibril formation).
The combination of ThT fluorescence with pyrene
fluorescence thus seems to reveal a remarkable two-step
aggregation process for A10H6, that is, the formation of
prefibrillar aggregates at micromolar concentration, followed
by a critical aggregation concentration associated with the
formation of more compact fibrils (vide infra) at a millimolar
peptide concentration.
We next examined the influence of gold nanoparticles on the
secondary structure of the peptide. The gold nanoparticles were
sized using several methods. UV/vis spectroscopy (Supporting
Information Figure 7) shows a plasmon absorption maximum
at 516 nm, which is consistent with 5 nm radius nano-
particles.39 From dynamic light scattering, a hydrodynamic
radius of 4.3 nm was obtained. TEM imaging (vide infra)
revealed a similar size. These values of particle size can be
considered to be the same within experimental uncertainties.
Spectroscopic methods all confirm that the addition of gold
NPs does not influence the secondary structure of the peptide.
This is shown through CD and FTIR spectroscopy, typical
results being shown in Figure 3. The CD spectra (Figure 3a)
show a positive peak near 200 nm with a negative band
Figure 1. Pyrene fluorescence assay to determine the critical
aggregation concentration in the micromolar range (cac1) of A10H6.
I1 and I3 are the fluorescence intensities measured at λem = 373 and
382 nm, respectively.
Pyrene fluorescence measurements led to a value for the cac1
of 0.0016 wt % (23 μM) at pH 7.6 as shown in Figure 1. The
fluorescence spectra from which the data reported in Figure 1
were obtained are reported in the Supporting Information
(Figure 4). The corresponding value for cac1 at pH 4.4 was 18
μM (Supporting Information Figure 5), suggesting that the
aggregation process leading to the formation of prefibrillar
aggregates is rather pH-independent, at least in this pH interval.
At much higher concentration, Thioflavin T spectroscopy,
supported by pyrene fluorescence measurements, reveals a
separate critical concentration (cac2) for fibril formation. This
is confirmed by the data in Figure 2. The two techniques reveal
a critical concentration of 0.5 wt % (ThT) and 0.16 wt %
(pyrene), corresponding to a cac2 = 2 mM. The difference in
values obtained from the two techniques may reflect the
distinct processes probed by these two dyes, that is, amyloid
formation in the case of ThT and hydrophobic sequestration in
the case of pyrene. The pH for the peptide around cac2 was
measured as pH 2.75 for an 0.5 wt % solution and pH 2.72 for a
1 wt % solution. These concentration for cac2 is much higher
than that observed for cac1 in the micromolar concentration
range (Figure 1). The former can tentatively be ascribed to the
formation of prefibrillar structures (vide infra). The local
change in polarity probed by pyrene fluorescence presumably
corresponds to the sequestration of the hydrophobic alanine
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reveals the presence of short fibrils (variable length, but not
longer than 100 nm), with a diameter of 5−10 nm. Figure 5
Figure 5. SAXS profile from a 0.5 wt % solution of A10H6 (open
circles), along with core−shell cylinder form factor model fit (red
line).
shows the SAXS intensity profile for a 0.5 wt % solution of
A10H6. The scattering data can be fitted to the form factor of a
core−shell cylinder,48 corresponding to cylinders with a dense
alanine-rich core and a hydrated histidine-rich corona. The
fitted fibril radius was 3.7 nm (with 50% Gaussian
polydispersity), consistent with the cryo-TEM image. The
SAXS data were fitted to the long core−shell cylinder model in
SASfit.48 The other fitting parameters for the results in Figure 5
are shell thickness ΔR = 0.1 nm and scattering contrast of core
η(core) = −0.0075, η(shell) = 0.161, and η(solvent) = 0.0035.
A flat background BG = 0.17 was included in the fit.
AFM (Supporting Information Figure 8) shows that, above
cac1, but below cac2, A10H6 self-assembles mainly into globular
prefibrillar structures; however, some fibrils are also present.
The latter appear to be formed by stacks of nanometer-scale
globules, suggesting that the cac2 transition is determined by a
further structural rearrangement leading to mature “amyloid-
like” fibrils.
X-ray diffraction also confirms a tightly packed β-sheet
structure for A10H6 in the fibrillar state above cac2. The results
shown in Figure 6 show strong peaks, corresponding to d
spacings of 4.42 and 5.34 Å. As for similar alanine-rich peptides
such as A6R49 and A6K,50 the former spacing is assigned as the
β-strand spacing, whereas the latter is the β-sheet spacing which
reflects the very close packing of β-sheets in oligo-alanine
peptides.
Figure 3. (a) CD spectra from 0.5 wt % solution of A10H6 without
(solid black line) and with (dashed red line) Nanogold. (b) FTIR
spectra (in deuterated buffer) of 3 wt % solution of A10H6 without
(solid black line) and with (dashed red line) Nanogold.
centered at 225 nm. These features are consistent with β-sheet
secondary structure.40−42 The position of the minimum in the
spectra is red-shifted from the usual location (216 nm) for β-
sheet structures, most probably due to light scattering resulting
from fibril aggregation.43 The FTIR spectra in the amide I′
region shown in Figure 3b contain peaks at 1631 and 1672
cm−1. The former peak is assigned to β-sheet structure,41,44
while the latter is due to TFA counterion binding to the
peptide.45−47
The formation of fibrils by A10H6 above cac2 is confirmed by
SAXS and TEM. Figure 4 shows a cryo-TEM image which
Figure 6. XRD profile obtained by integration of a fiber diffraction
Figure 4. Cryo-TEM image from a 1 wt % solution of A10H6.
pattern obtained from a stalk dried from a 1 wt % solution of A10H6.
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Since spectroscopic methods suggest that the Nanogold
particles do not influence the secondary structure of A10H6, we
next examined the influence of the nanoparticles on the self-
assembled fibril structures and potential labeling of the fibrils by
Nanogold. Figure 7 presents TEM and cryo-TEM images, both
Figure 7. Electron micrographs showing interaction of Nanogold with
A10H6 fibrils. (a) TEM image of 1.5 wt % A10H6 + 0.5 μM Nanogold in
MOPS. (b) Cryo-TEM image of 1 wt % A10H6 + 0.5 μM Nanogold in
MOPS.
of which clearly show the association of the Nanogold with the
peptide fibrils. The A10H6 nanostructures are short, disperse
fibrils resembling those for the peptide in the absence of
Nanogold (Figure 4). This again suggests association of the
gold nanoparticles with the peptide fibrils without disruption of
morphology (or secondary structure, probed by spectroscopic
methods). AFM also shows association of Nanogold with
peptide fibrils, a representative image being presented in Figure
8. From the line cross-section scans, it can be estimated that the
peptide fibrils are just under 6 nm in height, and the total
height of the nanoparticles on top of the peptide is 11−12 nm.
This suggests some compression of the underlying peptide
when the diameter of the Nanogold particles, around 8−10 nm,
is considered. The concentration of gold nanoparticles is low,
so that not all fibrils were observed to be decorated with them.
Remarkably, evidence for the binding of the Nanogold to the
histidine residues in the peptide could be obtained from CD
spectroscopy in the range 510−530 nm, that is, in the region of
the gold nanoparticle plasmon band (Supporting Information
Figure 9). The CD spectrum in Figure 9 shows a substantial
decrease of the negative minimum in this range by adding
micromolar A10H6 aliquots to Nanogold. The CD signal from
the functionalized gold nanoparticles is ascribed to an induced
Cotton effect caused by the chiral center on the NTA chain.
The linking of, for example, thiols causes a reconstruction of
the gold surface with some degree of stereochemistry.51
However, D- and L-type reconstruction is equally probable, so
the result is again optical inactivity. However, when chiral
molecules are linked to the gold surface, the D/L parity is
removed, and the plasmon transition is optically active, gaining
a net CD signal as demonstrated for peptide-tethered gold
nanoparticles.52,53 As this region of the spectrum is sensitive to
the Ni2+/histidine interaction,54−56 these results clearly indicate
the binding of the Nanogold to the peptide.
Figure 8. Top: AFM image showing Gold NPs associated with short
A10H6 fibrils (0.5 wt % peptide concentration in MOPS). Bottom: The
corresponding lines indicate cross sections.
Figure 9. CD spectra showing interaction of histidine residues in
A10H6 with gold nanoparticles, in the region of the plasmon absorption
band (Supporting Information Figure 6).
observed for other amyloid-forming peptides.57 For example,
computer simulations on the amyloid β peptide fragment
Aβ(16−22) show an initial step corresponding to the
sequestration of hydrophobic residues within molten
oligomers.57−61 The second aggregation step corresponds to
the reorganization within oligomers resulting from interchain
hydrogen bonding which produces β-sheet assemblies. Hints
for a similar initial hydrophobic collapse into globular
oligomers followed by subsequent β-sheet formation were
obtained from experiments on the related fragment Aβ(16−
20).62 It is possible that a similar mechanism occurs for the
surfactant-like peptide studied herein.
DISCUSSION
■
The aggregation process for A10H6 occurs via two distinct
critical aggregation concentrations. We are not aware of prior
reports on two-stage assembly processes for surfactant-like
peptides. However, two-step aggregation processes have been
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Present Address
We have shown that the hexa-histidine coating of the fibrils
could be used to tag the amyloid assemblies using Ni/NTA-
functionalized gold nanoparticles (Nanogold). This selective
tagging was shown not to influence the formation of β-sheet
secondary structures formed above cac2. Intriguing effects on
the gold nanoparticle Plasmon absorbance spectrum due to
binding to the peptide were observed via CD spectroscopy in
the visible wavelength range and our results show that the
Nanogold particles are optically active, presumably due to the
effect of the capping ligands. Binding to the β-sheet forming
A10H6 peptide leads to a reduction in the negative plasmon-
associated CD signal. For helical peptides, the sign of this chiral
absorbance band depends in an odd−even fashion on the
sequence length.53 A detailed model for this is still lacking, also
for our observation of modulation of the chiral Plasmon CD
signal upon binding to a model β-sheet forming peptide.
Our work suggests that if hexa-histidine-tagged proteins
misfold into amyloid structures they may be tagged using Ni-
NTA gold nanoparticles. Peptide nanotubes have been coated
with gold nanoparticles, and these assemblies show two-
dimensional charge transport behavior within the quantum dot
arrays.63 Other applications of hybrid peptide/nanoparticle
structures in sensing (using peptides immobilized on gold
nanoparticles) have been discussed.64 Our hybrid peptide/
inorganic nanowire structures may also have applications where
one-dimensional patterning of nanoparticles is exploited.
†V.C.: National Physical Laboratory, Hampton Rd, Tedding-
ton, Middlesex TW11 0LW, U.K.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by EPSRC Grant EP/L020599/1.
S.K. is the recipient of an STFC Futures Studentship. I.W.H.
acknowledges the support of a Royal Society-Wolfson Research
Merit Award for work on peptide self-assembly. We are grateful
to the ESRF for the award of beamtime on beamline BM29
(ref. MX-1606) and to Adam Round for assistance with the
measurements. The synthesis activity was supported by ESMI
(European Soft Matter Infrastructure, proposal ref
S130200339).
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Corresponding Author
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