Chirality of amyloid suprastructures

Article abstract of DOI:10.1021/ja800328y

Amyloids are pathological fibrillar aggregates of proteins related to more than 20 different diseases. Amyloid fibers have a characteristic cross-β structure consisting of a series of β-strands extended perpendicular to the fiber axis and joined by hydrogen bonds parallel to the fiber direction. Fibril aggregation results in helical suprastructures. Here we used high-resolution SEM and cryo-SEM for the study of chirality in the amyloid suprastructure. We found that amyloids of Aβ_1-40 and hen lysozyme form at all hierarchical levels always and only left-handed helices. In contrast, amyloid fibers formed by the N-terminal sequence of serum amyloid A (SAA_1-12) consist of right-handed helices exclusively. Consistently, the peptide enantiomer, formed of (R)-aminoacids, aggregates exclusively in left-handed helices. We conclude that the opposite handedness of the SAA_1-12 amyloids is an intrinsic feature of the peptide structure. The left-handed chirality observed for the Aβ_1-40 and hen lysozyme amyloid suprastructures is consistent with the conventional β-sheet structural model. In contrast, the right-handedness observed in (all-S) SAA_1-12 fibers indicates that the cross-β structure of SAA_1-12 fibers is probably not formed of β-sheets. Whatever the answer to the dilemma of the right-handed helicity of SAA_1-12 amyloid fibers is, its existence shows that the supramolecular chirality of amyloid fibers is not only dictated by the molecular chirality of the component molecules but also by their structural organization. Copyright

Full text of DOI:10.1021/ja800328y

Published on Web 03/14/2008
Chirality of Amyloid Suprastructures
Noa Rubin,† Emanuel Perugia,‡ Michal Goldschmidt,‡ Mati Fridkin,‡ and Lia Addadi*,†
Department of Structural Biology and Department of Organic Chemistry, Weizmann Institute of Science,
RehoVot, Israel 76100
Received January 15, 2008; E-mail: lia.addadi@weizmann.ac.il
Understanding the chirality of polypeptide suprastructures is an
issue of fundamental importance, which has attracted the attention
of protein scientists since the first models of protein structures
emerged. Thus, it is experimentally well-established that (S)-
aminoacid1 â-sheets assume a conformation characterized by a left-
handed twist along the sheet.2 Helical suprastructures resulting from
association of several â-sheets should subsequently also display
left-handed helicity. This assumption has far-reaching consequences
in the understanding of amyloid structures, where it has been both
experimentally confirmed and theoretically justified.3 Here we report
that in contrast to the common knowledge, the SAA1-12 peptide
consistently and exclusively forms amyloid fibers with right-handed
helicity at all hierarchical levels from 8 nm to tens of nanometers
width.
Amyloids are fibrillar aggregates of proteins with a characteristic
cross-â conformation.4 Over 20 diseases have been identified as
amyloid-related.5 The cross-â structure is minimally defined as a
series of â-strands extended perpendicular to the fiber axis and
joined by hydrogen bonds parallel to the fiber direction.4 Several
structures, all consistent with the cross-â definition, have been
however proposed for specific amyloids, such as â-sheet, â-helix,
or other â-folds.6-8 There is no consensus on whether each or all
of these models may be applicable in general or in specific cases.
The mature amyloid fiber consists of several protofilaments, with
widths ranging from 8 to 50 nm depending on the number of
protofilaments. The morphology of the fiber suprastructure may
vary between ribbons and helices even within the same sample.6,9
We have examined three disease-related amyloids: Aâ1-40, hen
lysozyme, and SAA1-12. Aâ1-40 is related to the Alzheimer disease.
The hen lysozyme protein is highly homologous to human lysozyme
which is related to systemic amyloidosis. SAA1-12 is a 12 amino
acid peptide (RSFFSFLGEAFD) from the N-terminal of the serum
amyloid A protein, which is related to secondary systemic amy-
loidosis (AA). It is commonly accepted that the amyloidogenic core
resides in the first 10-15 N-terminal amino acids of the protein.10,11
Amyloid fibers were induced to deposit from the three types of
peptides11,12 and characterized by transmission electron microscopy
(TEM) and ThT assay; the â-conformation of SAA1-12 amyloids
was verified by FTIR (see Supporting Information).
cryo-SEM allows observation at low temperature of biological
materials in hydrated form, without the need for fixation or staining
(Figure 1g-i). Here we used SEM and cryo-SEM for the deter-
mination of fiber handedness. We visualized ∼100 fibers from at
least three different fiber batches of each peptide.
We found that amyloids of Aâ1-40 and of hen lysozyme always
and only form left-handed helical fibers (Figure 1d and e,
respectively). Surprisingly, amyloids formed by the SAA1-12 peptide
always and only form right-handed helices (Figure 1f).
To eliminate the possibility that either dehydration or the fixation
procedure cause the right-handed chirality of the SAA1-12 fibers,
we performed cryo-SEM on freeze-dried samples of Aâ1-40 and
SAA1-12 amyloids. The observed chirality of both amyloid types
did not change, while the resolution improved (Figure 1g and h,
respectively). In order to confirm that the right-handedness of the
(all-S) SAA1-12 amyloids does not result from an artifact induced
by external parameters,13 we synthesized the peptide enantiomer,
(all-R) SAA1-12. The helicity of (all-R) SAA1-12 amyloids was
found to be exclusively left-handed, the mirror image of the (all-
S) SAA1-12 amyloids (Figure 1i).
All amyloids display both in TEM and SEM a wide range of
fiber widths, related to increasing hierarchical levels of aggregation
(Figure 2). The TEM clearly shows that the fibers are helical even
at the lowest hierarchy (8 nm). The SEM micrographs show that
the handedness of the fibers is consistent through all the hierarchy
levels of the different amyloid fibers (Figure 2a,d).
From all the above data, we conclude that the opposite handed-
ness of the SAA1-12 fibers, relative to the Aâ1-40 and to the hen
lysozyme fibers, is an intrinsic feature of the peptide structure. This
appears to indicate that the cross-â structure of SAA1-12 fibers is
not formed of â-sheets.
The left-handed chirality observed in the Aâ1-40 and hen
lysozyme amyloid suprastructures is consistent with the conven-
tional â-sheet structural model (Figure 2c). Indeed, (all-S) Aâ1-40
amyloids were shown by AFM to consist of left-handed helices,
while the enantiomer peptide amyloid fibers are right-handed.14 This
is further confirmed by a model based on ssNMR including the
assembly of four â-sheets in a protofilament.8 Similarly, in
agreement with our observations, AFM studies on human wild-
type lysozyme showed left-handed helical suprastructure.15
In contrast to Aâ1-40 and hen lysozyme, the right-handedness
observed in (all-S) SAA1-12 fibers and the left handedness of the
enantiomeric (all-R) peptide are not consistent with the conventional
â-sheet structural model. Although we cannot completely rule out
supercoiling as a possibility, we do not deem that it is the probable
cause for the right-handed helicity of the fibers. This is because
the same handedness was observed at all hierarchical levels, starting
with 8 nm fibers and up to 32 nm fibers.
Conventionally, amyloid morphology is determined by TEM after
negative staining. These images by definition do not contain
information on the handedness of fiber helices because TEM
micrographs provide projection images that cannot report on the
3-D morphology of objects (Figure 1a,b). TEM micrographs after
Pt shadowing may supply 3-D information on handedness only if
the grid orientation is known. In contrast, high-resolution scanning
electron microscopy (SEM), as well as AFM and STM, is suitable
for the determination of helix handedness because it directly
provides three-dimensional images (Figure 1c-f). Furthermore,
In a recent study, two diastereomeric amyloid populations were
shown to coexist.16 However, the system reached equilibrium at
higher temperature with only one fiber handedness, left-handed.
† Department of Structural Biology.
‡ Department of Organic Chemistry.
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10.1021/ja800328y CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. (a) Schematic representation of two helical fibers of opposite handedness (in color) and their projection images (gray). (b) Negative stain TEM
and (c) SEM micrographs of hen lysozyme. (d-f) SEM micrographs of (d) Aâ1-40, (e) hen lysozyme, and (f) (all-S) SAA1-12. (g-i) Cryo-SEM micrographs
of (g) Aâ1-40, (h) (all-S) SAA1-12, and (i) (all-R) SAA1-12. The resolution in (b) and (c) is ∼10 nm, and in (d)-(i) is ∼5 nm. The fibers observed in
cryo-SEM appear thicker because they are hydrated.
were found to form both left- and right-handed helical suprastruc-
tures.17 Examples of this same absence of unequivocal correlation
between molecular and supramolecular structures are also well-
documented for phospholipids, peptide amphiphiles, cellulose, and
DNA.18 Surprisingly, this is not documented and even less
understood for amyloid fibers.
Acknowledgment. We thank E. Shimony, E. Klein, I. So-
lomonov, D. Noy, S. Rubinraut, A. Kapitkovsky, and especially
E. Sone and A. Weiner for help and discussions. We thank the
Helen & Milton A. Kimmelman Center for support. L.A. is the
incumbent of the Dorothy and Patrick E. Gorman Chair, and M.F.
of the Lester B. Pearson Professorial Chair.
Supporting Information Available: SAA peptide syntheses,
amyloid preparation and characterization, EM sample preparation and
a complete reference list is available This material is available free of
charge via the Internet at http://pubs.acs.org.
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Figure 2. (a) SEM micrograph and (b) negative stain TEM micrograph of
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In contrast, for SAA1-12, the fiber helicity is consistently and
exclusively right-handed, showing that this is the most stable
conformation.
Whatever the answer to the dilemma of the right-handed helicity
of SAA1-12 amyloid fibers is, its existence shows that the
supramolecular chirality of (all-S) aggregated amyloid fibers may
be opposite, depending on their structural organization. This should
not be surprising, when considering that â-helices of (S)-aminoacids
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