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Fibrillisation of Ring-Closed Amyloid Peptides
Ian W. Hamley, *a Ge Cheng, a,b Valeria Castelletto, a Stephan Handschin,c Raffaele Mezzengac
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Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
First published on the web Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x
Ring-closing olefin metathesis reactions are used to create intramolecularly ring closed peptides or inter-molecularly ring-closed
peptide dimers based on a designed amyloid peptide sequence.
The uncrosslinked peptide self-assembles into high aspect ratio
nanotubes, however ring-closing leads to the formation of
fibrillar and twisted/helical ribbon structures.
The stabilization of peptide conformation is a major challenge
in the development of peptide therapeutics. Peptides offer great
potential in targeted delivery, however their stability against
proteolysis often needs improving. A number of approaches have
been developed to stabilize peptide and protein conformation
including stapling methods in which intra-molecular cross-linking
reactions are used to cross-link peptides or sequences within
proteins.1 For example, Grubbs and coworkers used ring-closing
olefin metathesis to cross-link amino acid side chains to produce
macrocylic helical peptides based on model hydrophobic
sequences.2, 3 The -helix within peptides based on a key domain
of the p53 tumor suppressor protein has also been stapled using
olefin metathesis methods,4 as have proteins involved in the
regulation of apoptosis5 and peptides designed to bind at the
coactivator binding site of the estrogen receptor.6 Other methods
have also been used to covalently cross-link peptides including
the application of the “click” copper-catalysed alkyne-azide
cycloaddition reaction to construct stabilized helices7, 8 and sheets.9 These reactions may also conveniently be used to prepare
cyclic peptides.10 Here, we report on the olefin-metathesis crosslinking of designed amyloid peptides which self-assemble into
nanotubes that comprise wrapped -sheet walls. The crosslinking reaction leads to intra-molecularly ring closed peptides, or
inter-molecularly ring-closed dimers. Despite the constraint
imposed by the architecture of the cross-linked fibrils, -sheet
fibril formation is still observed. The width of these objects is
controlled by the nature of the ring-closing process. We are not
aware of prior studies on the fibrillisation of ring-closed peptides,
and this is discussed here for the first time.
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Scheme 1. Structures of peptide I and alkene-bridged peptides II, III.
This journal is © The Royal Society of Chemistry [year]
The design of the peptide containing two allyl- functionalized
aspartic acid residues, D(All) is shown in Scheme 1. The peptide
was designed based on the KLVFFAE sequence A(16-22) from
the amyloid beta peptide.11-13 This contains the core KLVFF
sequence in amyloid fibrillisation and has been exploited as the
basis for a series of designed peptides under investigation in our
group.14-18
The
sequence
was
modified
to
EAFFD(All)D(All)VVLK, peptide I. The use of the allylfunctionalized glutamic acid residues, D(All), as cross-linkable
residues, was motivated by the commercial availability of the
corresponding Fmoc-D(All) amino acid. Peptide Ac-KLVFFAENH2 has been shown to self-assemble into nanotubes in pH 2
acetonitrile/H2O solutions.12, 19 Incorporating the two allyl ester
aspartic acid residues as well as an additional Val residue in
peptide I (also with a reverse sequence) still leads to nanotube
formation, and we were therefore able, uniquely, to investigate
the influence of metathesis ring-closing on peptide nanotube selfassembly.
The peptide E(OtBu)AFFD(All)D(All)VVLK(Boc) containing
cross-linkable allyl-functionalized aspartic acid residues
[D(OAll)] was prepared by solid phase peptide synthesis
methods. Deprotection afforded peptide I. Cross-linking reactions
were performed with the peptide on resin20, 21 using an olefin
metathesis reaction using a second generation Hoyveda-Grubbs
catalyst as described in the Supporting Information. Two
products, peptides II and III were obtained from this peptide.
Peptide II is an intra-molecularly ring-closed peptide, while III is
a ring-closed peptide dimer. In addition, byproducts associated
with the peptide I lacking the A residue were also identified and
labelled IV, V. These are discussed further in the Supporting
Information.
In the following, we investigate the self-assembly of II and III
compared to the uncross-linked peptide I using cryogenic
transmission electron microscopy (cryo-TEM) and small angle xrays scattering (SAXS) to probe the nanostructure in aqueous
solution; X-ray diffraction is further employed to elucidate details
of the molecular packing. Unexpectedly, despite the steric
constraints induced by ring-closing, the formation of amyloid
fibrils is observed for II and III.
The self-assembly of peptide I into nanotubes in aqueous
solution was first revealed by cryo-TEM and SAXS. Fig.1a
shows a cryo-TEM image which reveals tube-like structures with
a radius (25 4) nm. The tube walls appear to be thin, comprising
10% of the tube diameter. A nanotube structure was confirmed
by SAXS. Fig.1b shows the SAXS intensity profile, measured
over an extended q range. The intensity profile at low q contains
oscillations from the form factor of nanotubes. The red line
shows a fit to an approximate form factor of nanotubes with
infinitely thin walls, and with a radius of 22 nm, and a Gaussian
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polydispersity  = 10%. This form factor captures the location of
the form factor minima. Further detailed modeling is prohibited
by the presence of an additional contribution to the scattering
from structure factor at low q (giving rise to a broad intensity
maximum).
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data. In contrast to the data shown in Fig.1, nanotube form factor
oscillations are not observed for solutions of either peptide II or
III (Fig.3). These intensity profiles result from fibril selfassembly – the form factor maxima at q = 0.6 nm-1 (peptide II) or
q = 1.4 nm-1 (peptide III) are associated with the diameters of the
fibrils shown in Fig.2.
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(c)
q / nm-1
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Intensity
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Figure 1. Peptide I forms nanotubes in aqueous solution. (a) Cryo-TEM
image obtained from a 2.4 wt% solution, (b) Integrated SAXS intensity
profile (black points) and WAXS profile (blue points) along with
calculated low q form factor (red line) measured for a 2 wt% solution. A
similar profile was observed for an 0.5 wt% solution, (c) 2D-SAXS
pattern from the same 2 wt% sample, showing alignment of nanotubes
along the (horizontal) flow direction.
Figure 2. Cryo-TEM images. (a,b) Peptide II, 0.5 wt% in H2O, (c,d)
Peptide III, 0.25 wt% in H2O.
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The WAXS data obtained from the solutions is particularly
interesting. In the WAXS profile for peptide III shown in Fig.3,
peaks are observed at q = 5.6 nm-1 (d = 1.1 nm), q = 13.1 nm-1 (d
= 0.48 nm) and q = 16.5 nm-1 (shoulder, d = 0.38 nm). Peptide II
shows a broad shoulder at around q = 13.5 nm-1 (d = 0.47 nm).
Consistent with cryo-TEM, peptide II does not form welldeveloped -sheets at low concentration. However for peptide III
the in situ X-ray measurements, confirm the features of -sheet
ordering, i.e. the spacing of the sheets (1.1 nm) and the strand
spacing (0.47-0.48 nm) within the -sheets.
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The influence of cross-linking (intra-molecular in the case of
II, inter-molecular dimerization in the case of III) was examined
by cryo-TEM and SAXS. Fig.2 presents cryo-TEM images.
Peptide II does not self-assemble at 0.5 wt% (Fig. 2a,b).
However, at higher concentration (2 wt%) -sheet formation is
observed by FTIR (SI Fig.11) and fibrils are noted in cryo-TEM
images (SI Fig.12). In contrast, cryo-TEM indicates that Peptide
III forms a mixture of straight fibrils and shorter irregular
aggregates (Fig.2c,d) even at 0.5 wt%. This is supported by FTIR
(SI Fig.11) and negative stain TEM (SI Fig.13). For the fibrils,
the average diameter is (6.3 ± 0.5) nm. The cryo-TEM images
therefore reveal that neither II nor III form the nanotube structure
of the uncross-linked parent peptide I.
To complement real-space imaging, and to provide an in situ
probe of nanostructure over a larger volume than cryo-TEM,
SAXS (and WAXS) was performed. Fig.3 shows representative
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Intensity / mm
-1
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At high q, a series of Bragg peaks are observed that are due to
the configuration of the peptides within the nanotube walls (to be
discussed shortly). In addition, the WAXS profile measured
simultaneously (Fig.1b and expanded in SI Fig.10) contains peaks
assigned to the -sheet structure of the peptides, specifically
sharp peaks due to the -strand spacing (0.47 nm) and the -sheet
stacking distance (1.06 nm) as well as several additional
reflections. Spontaneous alignment of peptide nanotubes upon
transfer into the capillary flow-through cell used for SAXS was
observed, as revealed by the two-dimensional SAXS profile in
Fig.1c. This shows intensity maxima along the meridional
(vertical) direction, i.e. perpendicular to the horizontal flow
direction. These maxima are the primary form factor peaks in
Fig.1b.
Intensity
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Peptide II, 0.5% in H2O
Peptide III, 0.5% in H2O
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Figure 3. SAXS (and selected WAXS) data for peptide II and III in 0.5
wt% aqueous solutions. The inset shows an enlargement of the WAXS
profiles (in box) with linear intensity and q scales.
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To further investigate nanostructure, X-ray diffraction was
performed on dried stalks. Representative images are shown in SI
Fig.14. SI Fig.15 shows typical intensity profiles from equatorial
sector averages for peptide I and peptide II. This highlights
certain features associated with the nanotube structure of peptide
I, in particular the presence of peaks at d = 0.55 nm and d = 0.45
nm which are absent for peptide II, the XRD pattern for which
This journal is © The Royal Society of Chemistry [year]
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shows classical “cross-” pattern features, i.e. the 1.1 nm and
0.48 nm spacings (the same features were observed for peptide
III, data not shown). The peaks observed for peptide I were
previously observed in oriented X-ray diffraction patterns
(obtained by flow alignment) for the heptapeptide A6K which
forms nanotubes in concentrated aqueous solution.22 A model for
the origin of these peaks was presented based on the helical
wrapping of peptide dimers (with a two-residue offset between
adjacent dimers). An additional feature noted in the fibre XRD is
a d = 1.8 nm peak for both peptides. For peptide I, it may be
noted that a strong meridional reflection presumably associated
with the -strand spacing, is shifted such that the spacing is larger
than usual, d = 0.49 nm. The d = 0.45 nm peak is only present as
a shoulder. The d = 0.39 nm peak for peptide I is sharper and
more intense than the corresponding d = 0.38 nm peak observed
for peptide II. This peak is associated with the C – Cbackbone
spacing. 22
Molecular modeling (Cerius Molecular Dynamics using a
DREIDING force field) suggests a favored hairpin conformation
for peptide II, with a length approximately 1.8 nm (Fig. 4a). This
is in excellent agreement with the d spacing observed in the fibre
XRD pattern. The thickness of fibrils observed by cryo-TEM
suggests a width of three strands. For peptide III, molecular
dynamics simulations lead to a conformation (Fig. 4b) with a
planar arrangement in the cross-linked region, and with a longest
dimension approximately 3.5 – 4 nm. Detailed modeling of the
aggregation of the peptides into -sheet fibrils will be the subject
of future work. The length of the decapeptide I in an antiparallel
-sheet configuration can be estimated23 as 3.4 nm. The observed
d = 1.8 nm peak may be a second order reflection since it is
highly unlikely this peptide is folded.
(a)
(b)
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This work was supported by EPSRC grants EP/F048114/1 and
EP/G026203/1 to IWH. We are grateful to Dr T. Narayanan
(ESRF, Grenoble, France) for assistance with SAXS experiments.
We thank Prof Howard Colquhoun (University of Reading) for
assistance with the molecular modelling. Use of the Chemical
Analysis Facility at the University of Reading and of the Electron
Microscopy Facility of ETH Zurich (EMEZ) is acknowledged.
Notes and references
a
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Dept of Chemistry, University of Reading, Whiteknights Reading RG6
6AD, UK: E-mail: I.W.Hamley@reading.ac.uk
b
Present address: Dept of Chemistry, University of Liverpool, Crown
Street, Liverpool L69 3BX
c
Food & Soft Materials Science, Institute of Food Nutrition & Health,
ETH Zürich, LFO23 Schmelzbergstrasse 9, 8092 Zürich, Switzerland.
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† Electronic Supplementary Information (ESI) available: [Experimental
Methods and characterization data. Additional XRD data]. See
DOI: 10.1039/b000000x/
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Figure 4. Simulated conformations for (a) Peptide II, (b) Peptide III.
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In summary, ring-closing metathesis can be used to create an
intra-molecularly folded U-shaped peptide, whereas intermolecular ring-closing leads to extended four-arm “branched”
peptide dimers. The latter resembles in some respects a cyclic
peptide, although with a different architecture (e.g. much larger
branches than the side chains on cyclic peptides) and with a
distinct synthesis procedure. Regions of these peptides must
retain the -sheet hydrogen bonding pattern, consistent with the
XRD results presented. The conformational constraints
introduced by ring-closing hinder nanotube formation, observed
for the parent uncross-linked peptide. Instead, distinct fibrillar
and twisted/helical ribbon self-assembled nanostructures are
observed. The thickness of the fibril and ribbon structures is
related to the length of the peptides, whether intra- or intermolecularly ring-closed. Our results point towards the use of
ring-closing chemistries to create novel peptide architectures,
which are remarkably still able to form -sheet secondary
structures.
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