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Cite this: DOI: 10.1039/c0xx00000x
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Fibrils and nanotubes assembled from a modified amyloid- peptide
fragment differ in the packing of the same -sheet building blocks
Jillian Madine,a Hannah A. Davies,a Christopher Shaw,a Ian W. Hamleyb and David A. Middleton*a
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
The peptide AAKLVFF self-assembles into fibrils in water
and nanotubes in methanol. Solid-state NMR reveals that the
fibrils comprise -sheet bilayers whereas the nanotube wall
comprises monolayers or partially interdigitated bilayers.
Remarkably distinct morphologies are thus traced to subtle
differences in the arrangement of -sheet layers.
Many proteins and peptides assemble spontaneously into
nanoscale fibrillar structures characterised by the amyloid cross-
structure of repeating arrays of hydrogen bonded -strands 1.
Such assemblies are of great interest because of the role of
amyloid in diseases such as Alzheimer’s, type 2 diabetes and
many others 2. The conditions under which peptide self-assembly
occurs may be manipulated to produce novel, nonfibrillar
biomaterials with interesting morphological, tinctorial or
electronic properties 3, 4.
The peptide AAKLVFF, corresponding to residues 16-20 of
the A polypeptide extended at the N-terminus by two alanine
residues, forms well-ordered amyloid-like fibrils in water5 and
highly regular nanotube structures in methanol6, 7. Current data
suggest that the fibrils and nanotubes have the same cross-
structure but that the two morphologies differ in the spatial
arrangement of the inter-strand hydrogen bonds. Solid-state
nuclear magnetic resonance (SSNMR) methods are used here to
identify differences in the molecular arrangements within
AAKLVFF fibrils and nanotubes which underlie their nanoscale
morphologies. SSNMR has provided site-specific structural
information regarding the packing of short peptide structures and
for investigating differences in the packing arrangements of
polymorphic variants of assemblies from the same peptide 8-12.
Transmission electron microscopy (TEM) images of AAKLVFF
aggregates present in water and methanol after 5 d clearly
delineate slender fibrillar structures in water (Figure 1a) and
nanotubes in methanol (Figure 1b). The tubes are approximately
100 nm in diameter and over 1 μm in length and are deposited
more densely than the fibrillar structures. FTIR spectra of the
fibrils and tubes (Figure 1, c and d) show in both cases strong
bands at 1625 cm-1 and 1685 cm-1, which are together consistent
with antiparallel -strands6. The additional band at 1672 cm-1 is
assigned to the trifluoroacetate counterions of the two –NH3+
groups and the band at 1590 cm-1 is assigned to the 13C labelled
sites. The 13C cross-polarization magic angle spinning CP-MAS
SSNMR spectra of [13C2-AV]AAKLVFF fibrils and nanotubes
(Figure 1, e and f) show single peaks from the Ala2 methyl group
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Figure 1. Initial characterization of [13C2-AV]AAKLVFF fibrils (labelled
peptide nomenclature defined in the Supplementary Information)
assembled in water (left) and nanotubes assembled in methanol (right) by:
(a, b) TEM, (c, d) FTIR spectroscopy and (e,f) 13C CP-MAS SSNMR
spectroscopy.
at ~19.7 ppm and the Val5 carbonyl at~170.0 ppm. Both peaks
are somewhat narrower for the nanotubes than for the fibrils,
suggesting that the nanotubes are structurally more highly
ordered than the fibrils.
Rotational resonance (RR) NMR was used to detect throughspace 13C-13C dipolar couplings between Ala2 and Val5 of
hydrogen-bonded peptide -strands in [13C2-AV]AAKLVFF
fibrils and nanotubes. The experiment measures the exchange of
Zeeman order, which can be detected for 13C-13C distances of less
than 6.5 Ǻ. Molecular modelling of AAKLVFF indicates that
this distance range can only be satisfied when peptide -strands
adopt an antiparallel arrangement, with intermolecular hydrogenbonds between Val5 and Ala2 (“VA”) or between Val5 and Lys3
(“VK”) (Figure 2a). In each case two different intermolecular
13C-13C distances (5.9/3.9 Ǻ for VA and 5.6/6.4 Ǻ for VK) are
possible along the repeating unit of the hydrogen-bonding axis.
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Figure 2. RR 13C SSNMR analysis of the -strand registration in [13C2AV]AAKLVFF fibrils and nanotubes. (a) Two models of antiparallel strands for which the intermolecular 13C-13C distance is less than 6.5 Ǻ.
The two inter-strand distances given for each model are based on a
repeating distance of 4.7 Ǻ along the hydrogen-bonding axis as
determined by X-ray fibre diffraction. (b) Magnetization exchange at n =
1 (filled circles) and n = 2 RR (open circles) for fibrils (left) and
nanotubes (right). Solid lines represent the closest fitting simulated
curves corresponding to the “VA” arrangement and dashed lines represent
the closest fitting curves for the “VK” arrangement.
Rotational resonance data for the two morphologies clearly show
the exchange of Zeeman order (Figure 2b), confirming that the strands are antiparallel. The data were compared with numerically
simulated exchange curves (at n = 1 and n = 2 RR) based on the
two pairs of distances for the VA and VK -strand registrations
ZQ
and a range of values of T2 spanning the reciprocal sum of the
line widths. The closest agreement is in both cases seen for
distances of 5.9/3.9 Ǻ, i.e., the VA registration. Even at the
ZQ
upper limit of T2
the curves for the VK arrangement deviate
considerably from the data. The different exchange behaviour for
the two morphologies is a consequence of the relatively short
T2ZQ for the fibrils and long T2ZQ for the nanotubes, which can
be traced to difference in the NMR line widths for the two forms
in Figure 1, e and f. The two morphologies thus share the same
-strand alignment, in which peptides are staggered such that the
terminal amino acids create an overhang (Figure 2a, left). A
consequence of the hydrogen bonding pattern for the VA
registration (Figure 2a) is that all 7 amino acid side-chains are
represented equally on both faces of the -sheet. (i.e., the
“face=back” -spine defined in Sawaya et al. 13). The peptide
KLVFFA (A16-21) also assembles into fibrils with a face=back
configuration with internal two-fold screw symmetry 14.
Further SSNMR methods were used to search for differences
in the molecular organization of the fibrils and nanotubes which
could give rise to their distinct morphologies. Recent SSNMR
measurements on nanotubes formed by another A-derived
peptide, KLVFFAL, showed that antiparallel -strands were
organized into a bilayer packing structure9. Here an SSNMR
experiment was conducted in which [13C-F]AAKLVFF nanotubes
and fibrils were titrated with a MnCl2 solution to investigate
whether a bilayer structure occurrs in the AAKLVFF assemblies.
Paramagnetic cation such as Mn2+ bind to negatively charged
groups (such as C-terminal carboxyl groups) on the accessible
surfaces of molecular assemblies and shorten the T2 relaxation
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Figure 3. SSNMR experiments to probe -sheet packing in AAKLVFF
assemblies. (a) The concentric circles (representing Mn2+) indicate how
the paramagnetic ion can access all C-terminal carboxyl groups in a
monolayer of antiparallel -strands. The arrows denote the distances
between 13C labels in the carboxyl groups. (b) Peak intensity for [ 13CF]AAKLVFF fibrils and nanotubes titrated with MnCl2 to molar
equivalence or higher. (c) 13C MQNMR analysis of -sheet packing.
times of the 13C spins in closest proximity to the ions 15, 16. The
enhancement of T2 relaxation can be seen as a [Mn2+]-dependent
decrease in the NMR peak intensity.
For a monolayer
arrangement all C-termini are exposed to Mn2+ on each face of
the monolayer, whereas for multilayer assemblies at least half of
the C-termini are buried at the interface between individual
monolayers and are therefore less accessible to Mn2+ (Figure 3a).
The spectra of the nanotubes titrated with Mn 2+ show a decrease
in the carboxyl peak intensity until virtually no signal remains at
an equimolar Mn2+:peptide ratio (Figure 3b, left and right). This
trend suggests that all C-termini are exposed and thus the data
favours a monolayer arrangement in the nanotubes. The spectra
of the fibrils also show a decrease in peak intensity, but no more
than 50 % of the signal is abolished even when Mn2+ is titrated to
a molar excess (Figure 3b, middle and right). This response is
consistent with a bilayer arrangement in the fibrils, in which half
of the C-termini are excluded from the paramagnetic ions.
Further evidence was gained from multiple-quantum (MQ)NMR
measurements on [13C-F]AAKLVFF nanotubes and fibrils (in the
absence of Mn2+). The coherence orders excited represent the
number of spins that are correlated through a dipolar-coupling
network (i.e., with 13C spins separated by < 6.5 Ǻ), and can report
on the supramolecular structure of fibrils by identifying clusters
of groups or molecules. Excitation of higher orders of coherence
indicates a greater number of spins in the dipole-dipole coupling
network. In a monolayer arrangement of antiparallel -strands,
the Phe7 13C spins are separated by ~ 10 Ǻ and are too far apart to
give rise to detectable coupling, whereas in a multilayer
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be a multiple of the -strand distance of 4.7-4.8 Ǻ) 6. Small angle
X-ray scattering analysis of the nanotubes determined the wall
thickness to be 35 Ǻ 7, which is less than the length of a true
bilayer but somewhat longer than expected for a monolayer.
Indeed, more recent work on AAAAAAK reports a single wall
peptide nanotube system for which there are some characteristic
features in the fibre X-ray diffraction data not observed for
AAKLVFF 17. We therefore propose an offset monolayer or
partially interdigitated bilayer arrangement for the nanotubes,
which satisfies the Mn2+ accessibility and MQNMR constraints
and the dimensions determined from the earlier data (Figure 4b).
In summary, this work demonstrates how remarkably distinct
morphologies can originate from the same -sheet building
blocks but organised in subtly different ways.
We thank Dr Valeria Castelletto for assistance with TEM.
Notes and references
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Figure 4. Models of AAKLVFF assemblies. (a) Space-filled model of
fibrils (top) and one possible peptide arrangement viewed along the fibril
axis a (bottom). The model arbitrarily comprises 4 -sheet bilayers, with
c being the bilayer axis. (b) Space-filled model of nanotubes (top) and a
slice through a single peptide layer (perpendicular to the hydrogen
bonding axis) illustrating the interdigitated bilayer arrangement (bottom).
arrangement the spins may be separated by as little as ~ 5 Ǻ
(Figure 3a). The MQNMR spectra of the nanotubes detected
only zero quantum signals (Figure 3c, left), indicating that no
13C-13C coupling occurs in this assembly. The MQNMR spectra
of the fibrils detected up to n = 6 orders of quanta, consistent with
an extended network of coupled 13C spins across the monolayermonolayer interface (Figure 3c, right). Nevertheless, the detection
of several orders of quanta is consistent with an arrangement with
repeat 13C spacings of less than 6.5 Ǻ. The two morphologies
therefore differ at the molecular level in the arrangement of the
same antiparallel -sheet building blocks.
Earlier fibre diffraction data for the fibrils provided an
orthorhombic unit cell with unit axes a = 9.6 Ǻ, b = 18.6 Ǻ and c
= 43.7 Ǻ 5, where a coincides approximately with the hydrogen
bonding axis, b is related to the spacing between -sheet layers
and c coincides with the long axis of the -strand. The cell axis
c corresponds approximately to two AAKLVFF -strands of
length 23.8 Ǻ and thus supports the bilayer configuration
proposed here. Figure 4a shows a model for the fibril structure
satisfying the SSNMR data and the unit cell dimensions. These
constraints are unable to elucidate unambiguously the relative
orientations of the individual -sheet layers along the b axis.
Here we suggest a steric zipper interface analogous to the
polymorphic forms I and III of KLVFFAL microcrystals14,
allowing  interactions between Phe side groups of adjacent
layers. Fibrils comprise three protofilaments of average width 21
 6 nm5, which corresponds to at least 10 bilayer repeats along
the -sheet axis b (Figure 4a shows only 4 repeats). Initial
interpretation of the NMR data for the nanotubes suggested a
monolayer arrangement of -sheets. Fibre diffraction data for the
nanotubes provided a unit cell with unit axes b = 19.2 Ǻ and c =
42.0 Ǻ (unit cell dimension a was not well-defined but is likely to
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Department of Structural and Chemical Biology, University of
Liverpool, Crown Street, Liverpool, U.K.. Fax: +44 151 795 4406; Tel:
+44 151 7954457; E-mail: middleda@liv.ac.uk
b
Department of Chemistry, University of Reading, Reading RG6 6AD,
U.K.
† Electronic Supplementary Information (ESI) available: Experimental
methods and materials. See DOI: 10.1039/b000000x/
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