COMMUNICATIONFibrils 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
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XXDOI: 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
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 through-space 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 hydrogen- bonds 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
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possible along the repeating unit of the hydrogen-bonding axis.
Figure 2. RR 13C SSNMR analysis of the -strand registration in [13C2-AV]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 and a range of values of T 2
ZQspanning 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 upper limit of T 2
ZQ 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 T 2
ZQ for the fibrils and long T 2
ZQ 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 (A16-21) 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
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 [13C-F]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 Mn2+ 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
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give rise to detectable coupling, whereas in a multilayer
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 monolayer-monolayer 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 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.
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