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Feature article
Amyloids: From molecular structure to mechanical properties
Michael Schleeger
a
, Corianne C. vandenAkker
b
, Tanja Deckert-Gaudig
c
, Volker Deckert
c
,
d
,
Krassimir P. Velikov
e
,
f
, Gijsje Koenderink
b
, Mischa Bonn
a
,
*
a
Max Planck Institute for Polymer Research, Department of Molecular Spectroscopy, Ackermannweg 10, 55128 Mainz, Germany
b
FOM Institute AMOLF, Science Park 104, 1098 XG, Amsterdam, The Netherlands
c
Institute of Photonic Technology, Albert-Einstein-Str. 9, 07745 Jena, Germany
d
Institute for Physical Chemistry & Abbe School of Photonics, University of Jena, Helmholtzweg 4, 07743 Jena, Germany
e
Unilever R&D Vlaardingen, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands
f
Soft Condensed Matter, Debye Institute for Nanomaterials Science, Department of Physics and Astronomy, Utrecht University, Princetonplein 5, 3584 CC Utrecht, The Netherlands
article info
Article history:
Received 9 November 2012
Received in revised form
21 January 2013
Accepted 11 February 2013
Available online xxx
Keywords:
Amyloids
Vibrational spectroscopy
Biopolymers
abstract
Many proteins of diverse sequence, structure and function self-assemble into morphologically similar
fibrillar aggregates known as amyloids. Amyloids are remarkable polymers in several respects. First of all,
amyloids can be formed from proteins with very different amino acid sequences; the common de-
nominator is that the individual proteins constituting the amyloid fold predominantly into a
b
-sheet
structure. Secondly, the formation of the fibril occurs through non-covalent interactions between pri-
marily the
b
-sheets, causing the monomers to stack into fibrils. The fibrils are remarkably robust,
considering that the monomers are bound non-covalently. Finally, a common characteristic of fibrils is
their unbranched, straight, fiber-like structure arising from the intertwining of the multiple
b
-sheet
filaments. These remarkably ordered and stable nanofibrils can be useful as building blocks for protein-
based functional materials, but they are also implicated in severe neurodegenerative diseases. The overall
aim of this article is to highlight recent efforts aimed at obtaining insights into amyloid proteins on
different length scales. Starting from molecular information on amyloids, single fibril properties and
mechanical properties of networks of fibrils are described. Specifically, we focus on the self-assembly of
amyloid protein fibrils composed of peptides and denatured model proteins, as well as the influence of
inhibitors of fibril formation. Additionally, we will demonstrate how the application of recently devel-
oped vibrational spectroscopic techniques has emerged as a powerful approach to gain spatially resolved
information on the structureefunction relation of amyloids. While spectroscopy provides information on
local molecular conformations and protein secondary structure, information on the single fibril level has
been developed by diverse microscopic techniques. The approaches to reveal basic mechanical properties
of single fibrils like bending rigidity, shear modulus, ultimate tensile strength and fracture behavior are
illustrated. Lastly, mechanics of networks of amyloid fibrils, typically forming viscoelastic gels are out-
lined, with a focus on (micro-) rheological properties. The resulting fundamental insights are essential for
the rational design of novel edible and biodegradable protein-based polymers, but also to devise ther-
apeutic strategies to combat amyloid assembly and accumulation during pathogenic disorders.
Ó2013 Elsevier Ltd. All rights reserved.
1. Introduction
Amyloids constitute a fascinating class of biopolymers that
consist of non-covalently bound aggregates of misfolded poly-
peptides, resulting in insoluble fibrous protein aggregates sharing
specific structural traits. Amyloids are set apart from other bio-
polymers such as DNA and polysaccharides, in that the respective
monomers of amyloids are comparatively large polypeptides which
polymerize to form a so-called cross-
b
structure. Moreover, the
bonds between the monomers are non-covalent. As a result of
misfolding, the individual polypeptides constituting an amyloid
fibril fold predominantly into a
b
-sheet structure. The strong, but
non-covalent interaction between the
b
-sheets gives rise to stack-
ing of the peptides into protofibrils, which can subsequently
assemble into large fibrils with a diameter of several nm’s and a
length up to many microns. Many different biological and artificial
peptides can form amyloid structures under the right conditions.
In vivo, amyloids are often associated with neurodegenerative dis-
eases like Alzheimer, diabetes and Parkinson’s disease. However
both biogenic and artificial amyloids are also widely applied, for
instance, in structuring foodstuffs.
*Corresponding author. Tel.: þ49 6131 3791 60; fax: þ49 6131 379 360.
E-mail address: bonn@mpip-mainz.mpg.de (M. Bonn).
Contents lists available at SciVerse ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
0032-3861/$ esee front matter Ó2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.polymer.2013.02.029
Polymer xxx (2013) 1e16
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dx.doi.org/10.1016/j.polymer.2013.02.029
Amyloid fibrils can be regarded as biopolymers very similar to
silk [1], exhibiting common key features: they originate primarily
from unstructured precursor proteins and the definition of the
materials occurs through its specific structural and mechanical
properties rather than through its detailed chemical composition.
The cross-
b
sheet core structure of amyloid fibrils is very rigid and
confers superior mechanical properties. The fibrils can exhibit a
Young’s modulus similar to that of silk [2] and an ultimate strength
similar to steel [2,3]. As such, amyloid fibrils constitute promising
building blocks for bio-inspired materials. The fibrils form spon-
taneously from a wide range of natural proteins. The structural and
mechanical properties are relatively insensitive to the protein
amino acid sequence [4] and to a wide range of chemical and
biochemical modifications [5]. The unique structure of amyloid fi-
brils renders them relatively robust, even under extreme conditions
such as high and low temperature, the presence of proteases, de-
tergents and denaturants, and physical forces [6]. The recent in-
crease in research activity in the area of amyloid-based materials
[7] is therefore not surprising. Amyloid fibrils have been used as
templates for metallic nanowires that could be used for molecular
electronics [6], have been proven to be efficient drug delivery ve-
hicles [8] and are useful as scaffolds for tissue engineering [9].In
the context of human diseases (see Section 2), their robustness
against chemicals generally impedes effective medical treatment of
amyloid-based diseases.
Despite the broad interest in this relatively novel class of ma-
terials, a detailed understanding of the relation between the mo-
lecular properties of amyloids and their material characteristics like
stiffness and mechanical strength has remained challenging. For
instance, the high
b
-sheet content as the main secondary structure
characteristic of amyloids is related to their structural stability [3].
But amyloids contain additional secondary structures and exhibit a
distinct polymorphism [10e12], seeming to weaken the exclusive
role of
b
-sheets as the determinant of amyloid stiffness and me-
chanical strength. In particular, the role of the amino acid side
chains remains to be elucidated in that context. The lag in our basic
understanding of the relation between amyloid structure and me-
chanical properties may be traced back to the limited number of
techniques that can provide information on the molecular in-
teractions underlying the assembly and material properties of
protein fibrils. The material properties of amyloids are based on
delicately interconnected effects occurring at a variety of length
scales, as illustrated in Fig. 1. Our article will highlight recent
applications of various techniques aimed at elucidating the mo-
lecular structures and mechanical properties of amyloid-fibrils and
derived materials.
Specifically, this feature article describes recent investigations
into the structural and mechanical properties of amyloids at
different length scales. Nanoscopic level insights are obtained with
recently developed vibrational spectroscopic techniques. The vi-
brations of molecular groups within amyloid structures, and in
particular the amide I mode (with predominant C]O stretch
character), are sensitive reporters of the local environment of those
groups, as well as the protein structure. Hence, vibrational spec-
troscopies are very useful for quantifying secondary structure and
intermolecular interactions in amyloid systems. Moreover, surface-
specific vibrational spectroscopies like vibrational sum frequency
generation (vSFG) can provide information about amyloid forma-
tion at (lipid-) interfaces, which are known to catalyze the poly-
merization of amyloid precursors. The application of tip-enhanced
Raman spectroscopy (TERS) offers the possibility to spatially
resolve structural characteristics of amyloids in the nm regime.
Two-dimensional infrared spectroscopy (2D-IR) reveals amyloid
interactions down to the level of single amino acids, by selective
isotopic substitution.
At the mesoscopic level, we review results from biophysical
assays using transmission electron-, atomic force and fluorescence
microscopy to measure single fibril mechanical properties. Among
others, several different approaches to determine values for
bending rigidity, shear modulus, ultimate tensile strength and
extensibility are presented. Rheology measurements have been
applied at the microscopic scale to access the mechanical proper-
ties of networks of fibrils, which constitute promising materials like
viscoelastic gels.
2. Background
Many biological materials rely on fibrous networks of proteins
for their mechanical strength. Some well-known examples include
tissues such as skin, blood clots, and spider webs. Proteins are
normally folded in a specific geometry dictated by their primary
structure (amino acid sequence). Fibrils are then formed by su-
pramolecular assembly of protein building blocks, which are often
globular (as in the case of actin and microtubules) or rod-like (as in
the case of collagen and fibrin blood clots).
There is also an alternate path of fibril formation: misfolded or
partially unfolded proteins tend to form amyloid fibrils. This
pathway was originally discovered in the context of several
neurodegenerative diseases (notably Alzheimer’s, Parkinson’s,
Huntington’s, and Prion disease) and late onset diabetes [13,14].
These ‘conformational diseases’(or amyloidoses) are characterized
by the deposition of insoluble plaques of aggregated amyloid fibrils,
which can lead to cell death in specific organs. Surprisingly, several
organisms use amyloids to build natural, protective materials, such
as protective coatings of bacteria and spores and the silkmoth
eggshell [15]. It is increasingly apparent that the amyloid state is an
inherent characteristic of polypeptide molecules under denaturing
conditions, independent of the native structure or primary
sequence. In the context of food, this has been long known, for
instance in the cases of heat-denatured gelation of
b
-lactoglobulin
from milk and lysozyme from egg white [4].
Amyloid fibrils formed from structurally unrelated peptides and
proteins share a surprisingly similar structure. The change in sec-
ondary structure of the native protein to a
b
-sheet rich secondary
structure represents one of the main determinants of amyloid
formation. Amyloid fibrils show a characteristic X-ray diffraction
pattern, caused by a so-called cross-
b
core structure. The amyloid
core structure is composed of a stack of
b
-strands perpendicular to
Fig. 1. Hierarchical length scales relevant in amyloid research, starting from the level
of single molecules (left side: hIAPP, PDB 2L86) in the nanoscopic regime. Single or
several peptides and proteins build up secondary structure elements, which are
dominated in amyloids by
b
-sheets (PDB 2KIB). Mature amyloid fibrils have meso-
scopic dimensions and consist typically of two or more twisted strands, forming thin
and long fibrils. Networks of fibrils constitute micro- and macroscopic materials.
Vibrational spectroscopies focus on insights at the molecule and secondary structure
element level, atomic force and electron microscopy (AFM and EM) at the level of
single fibrils and (micro-) rheology on networks of amyloids.
M. Schleeger et al. / Polymer xxx (2013) 1e162
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the fibril axis separated by 4.8
A and with an intersheet spacing in
the order of 9e11
A[16,17]. The elongated stack is stabilized by a
dense network of hydrogen bonds. The sequence-specific side-
chains tend to affect the propensity to form fibrils [18]. A model of a
protofibril from human amyloid polypeptide hIAPP is shown in
Fig. 2. A single hIAPP molecule forms two
b
-sheets, separated by a
loop region. The views along the entire fibril axis in the model
reveal the dense stacking of four
b
-strands from two individual
IAPP molecules per layer. This in-register packing of parallel
b
-sheets is one of the prominent characteristics of amyloids. A view
perpendicular to the fibril axis (Fig. 2D) illustrates the 4.8
A spacing
between the stacks of parallel
b
-sheets and their parallel orienta-
tion to the fibril axis. Two or more protofibrils usually twist
together to form unbranched, elongated mature amyloid fibrils
which look like twisted rope-like structures or flat tapes, depend-
ing on the protein Ref. [12]. The high degree of structural order
within fibrils leads to very strong interactions between the proto-
fibrils within a mature amyloid fibril (for instance 310 k
B
T/
m
m for
insulin Ref. [19]). The fibrils are typically about 10 nm in width
(with a range of 5e25 nm) and up to 10
m
m in length.
The unique molecular organization of amyloid fibrils endows
them with remarkable mechanical properties. Amyloid fibrils are
among the stiffest biological materials presently known, with a
Young’s modulus on the order of 3e20 GPa [4]. Moreover, amyloid
fibrils exhibit a high resistance to breakage. Their ultimate strength
was shown to be on the order of 0.6 GPa, comparable to the strength
of silk but also steel [2]. This large fracture strength seems surprising
since amyloid fibrils are held together by non-covalent interactions.
Recent experimental and theoretical work has demonstrated that
the remarkable rigidity of amyloid fibrils originates from the regular
network of intermolecular hydrogen bonds in the cross-
b
core [3].
Non-covalent interactions between the variable side-chains (hy-
drophobic or hydrogen-bonding) sometimes further stabilize the
fibril and enhance the Young’s modulus [3].
From a materials science perspective, the amyloid pathway to
form protein fibrils has many advantages. The fibrils readily self-
Fig. 2. Structural model of an amyloid protofibril from human islet amyloid polypeptide (hIAPP) based on the crystal structure from segments of the peptide. AeC View along the
fibril axis. A. Two hIAPP molecules, each consisting of a hairpin and a steric zipper interface, tending to the fibrils axis. The space filling model B. emphasizes the tight steric zipper
interface of the two IAPP molecules. C. Stacking of
b
-sheet segments which are coiled up around the fibril’s axis. D. View perpendicular to the fibril axis, revealing the typical 4.8
A
spacing between layers of stacked
b
-sheets. The width of the fibril is 64
A, the marked 125
A distance matches a quarter of a full helical turn. Reprinted with permission from Ref.
[17]. Copyright 2008 The Protein Society.
M. Schleeger et al. / Polymer xxx (2013) 1e16 3
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assemble from natural proteins including edible proteins (such as
b
-lactoglobulin) or pharmaceutically relevant proteins (such as
insulin [19,20]), but they can also be formed from simple de novo
designed peptides [21,22]. Fibrils can also be formed from proteins
or peptides fused with functional proteins or peptides such as en-
zymes or cell adhesion motifs [9,23]. The mechanical properties are
remarkably insensitive to the protein sequence [4], and robust
under extreme conditions, such as high temperature, high con-
centrations of detergents and denaturants, as well as strong phys-
ical forces [6]. All of this makes them attractive candidates for
applications. Indeed, there has recently been a surge of activity
directed at designing materials from amyloids [7]. Amyloid fibrils
have been coated with metal [6] and conducting polymers [24] to
form conducting nanowires. Since amyloids are biocompatible,
their use as drug delivery vehicles [8] or applications in tissue en-
gineering [9] are also highly promising.
However, the flipside of the remarkable stability of amyloid
aggregates is that such stability is undesirable during the produc-
tion and storage of pharmaceutical and industrial peptides [25].
More importantly, the stability is also particularly undesirable in
the context of conformational diseases, such as Alzheimer’s and
Parkinson’s disease and type II diabetes, where amyloid fibrils are
the cause of disease symptoms and accumulate in tissues because
they cannot be degraded. More than 20 proteins that are partially
unfolded, misfolded, or aggregated are known to give rise to am-
yloid fibrils in humans. There has been an intensive search for small
molecules and peptides that can inhibit the formation of fibrils
[14,26]. Originally, it was believed that the fibrils were the major
toxic species (the ‘amyloid hypothesis’). However, recently it has
become apparent that oligomeric species may be even more toxic
[27]. Thus, small molecule or peptide-based drugs should not only
inhibit fibrillization, but also divert polypeptide assembly down
alternative, less harmful assembly pathways [28].
Here we focus mainly (but not exclusively) on two exemplary
amyloids, the disease-related human amyloid polypeptide (hIAPP)
or amylin and the primarily food-related
b
-lactoglobulin (
b
-lg).
hIAPP forms extracellular deposits in the pancreas of type 2 dia-
betes mellitus patients [29]. hIAPP is in vivo cosecreted with insulin
by the
b
-islet cells of the pancreas. Amyloid fibrils and/or oligo-
meric forms of hIAPP are thought to induce cell death of islet cells,
eventually destroying insulin production [29e32].Forin vitro
studies, hIAPP is commonly prepared by solid-state synthesis, since
it is a short peptide of 37 amino acid residues. In contrast,
b
-lg is a
globular protein from milk that is of great interest for industrial
(food) applications [33]. Amyloid fibrils can be readily formed by
heat-induced denaturation at low pH [34e37]. Both hIAPP and
b
-lg
are examples for well-studied systems in terms of kinetics and
thermodynamics of formation (
b
-lg [36,38]; hIAPP [39]) and fibril
structure (
b
-lg [34,37]; hIAPP [17,40e42]).
Recently, plant polyphenols have drawn attention as a possible
candidate to control amyloid formation and stability. For instance,
polyphenols are promising as a drug for prevention or treatment of
Alzheimer’s disease [4,43,44], and affect the quality of food-related
materials [45]. They are ubiquitous compounds in plant-derived
food and beverages (e.g. green tea) and have recently been
shown to be potential inhibitors of amyloid fibrillization [43,44,46].
Polyphenols are thought to have many more beneficial properties
based on their antimicrobial properties and their antioxidative
capacities, including therapeutic potential for cancer [47] and car-
diovascular diseases [48]. The strong interactions between poly-
phenols and amyloid proteins could potentially be used to combat
amyloidosis, but proteins could also serve as delivery vehicles of
polyphenols in the form of polyphenol-rich food or of drug delivery
agents. Polyphenols are thought to interact with amyloids and
amyloid precursor proteins via aromatic residues [49e51].
3. The nanoscopic level espectroscopic insights into the
molecular structure of amyloids
Vibrational spectroscopy is being widely applied for structural
analysis of biopolymers. In this article, we will refrain from a
description of classical Raman and FT-IR spectroscopy, despite the
significant importance of these techniques in current amyloid
research [52,53]. We will focus on “new techniques”with only a
very brief and fragmentary description of the techniques them-
selves, rather emphasizing the new insights into amyloid fibrils
provided by such techniques with the use of a few significant
examples.
The development of two relatively new vibrational techniques,
coherent two-dimensional infrared spectroscopy (2DIR) and
vibrational sum frequency generation spectroscopy (vSFG) have
depended strongly on the development and common availability of
infrared laser systems with ultra-short pulse duration. Their
application to biopolymers is still at its beginning, not only
providing the general structural sensitivity of vibrational spec-
troscopy but further offering an intrinsic possibility to perform
ultrafast time-resolved experiments down to femtoseconds, the
time domain of formation and breaking of chemical bonds [54].
Tip-enhanced Raman scattering (TERS) provides another route to-
wards the investigation of nanoscale structural variations, utilizing
plasmonic particles for sensitivity enhancement and spatial reso-
lution improvement [55,56]. TERS enables even the investigation of
the polymorphism of single amyloid fibrils [57]. In the following,
recent results obtained with these three vibrational spectroscopic
approaches are highlighted.
3.1. Vibrational sum frequency generation spectroscopy (vSFG)
Vibrational sum frequency generation spectroscopy (vSFG)
emerged after the development of ultrafast infrared lasers as a
technique mostly applied under ultra-high vacuum (UHV) condi-
tions to characterize the vibrational spectra of molecules adsorbed
onto surfaces. Nowadays vSFG is routinely applied to complex
biologically relevant samples, with its rather unique surface spec-
ificity, combined with high sensitivity and time resolution
[54,58,59]. The main strength of vSFG is that it allows one to record
the vibrational spectrum of specifically the outermost monolayer of
a bulk material, with high sensitivity.
In a typical vSFG experiment, ultrashort amplified laser light
pulses (e.g. at a wavelength of 800 nm) are split to allow for the
subsequent generation of spectrally narrow visible and broadband
mid-infrared laser pulses. The two coherent pulses are spatially and
temporally overlapped onto the sample surface and interact with
the interface by generating light of the sum frequencyof the visible
and mid-infrared light. This process is enhanced if the infrared light
is in resonance with vibrational modes on the surface. Using
spectrally resolved detection, the frequencies of surface vibrations
are readily extracted from the vSFG spectra by subtracting the
frequency of the narrowband visible pulse. The resulting vSFG
spectrum is usually normalized to the non-resonant vSFG signal of
a reference material or the intensity spectrum of the broadband IR
laser pulse (for a detailed review we refer to [54]). Heterodyne
detection may be applied to obtain not only intensity, but also
amplitude and phase information of the spectra, an approach
which is helpful in particular if vibrational modes strongly overlap.
In addition to its sensitivity, the specificity of vSFG to non-
isotropic samples offers the remarkable possibility to measure
structurally sensitive vibrational spectra exclusively at interfaces.
Isotropic bulk media show no SFG spectra due to the selection rules
of the underlying second-order nonlinear optical process. The
second-order polarizability, the source term of the SFG light, is zero
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for any medium with inversion symmetry. At interfaces, the sym-
metry of the centrosymmetric bulk phase is broken, allowing for
measurements of vibrational spectra specifically at the interface.
The sensitivity of vSFG to surface monolayers enables for
instance the investigation of the very same amyloid samples which
are suitable for atomic force microscopy (AFM) as well. Therefore a
molecular method can be directly aligned to a mesoscopic method.
This approach can be applied to consecutively analyze secondary
structure and e.g. stiffness parameters of amyloids grown at
different conditions. The spatial resolution of a typical SFG setup is
restricted by the size of the two overlapping laser pulses. Typically,
the SFG signal is averaged over an area somewhat less than one
square millimeter. The application of AFM and SFG together allows
therefore the combination of a high spatial resolution technique
with a surface sensitive label-free vibrational spectroscopy.
The combination of vSFG and tapping mode AFM was used to
characterize different morphology forms of amyloids grown from
b
-lactoglobulin [60]. It was shown that
b
-lg forms small, worm-like
amyloids when grown under high concentration (7.5% w/v) con-
ditions and long, straight fibrils at lower protein concentration
(3% w/v) [60]. The stiffness of the two morphologically different
amyloid forms differs strongly (see below), even though they
display the same fibril diameter. vSFG was applied to reveal the
molecular origin of this surprising behavior [60].InFig. 3a, vSFG
spectra in the amide I spectral region of amyloid fibrils obtained
from different growth conditions are presented. The position of the
vSFG band enables the determination of the secondary structure
distribution of the samples. Fig. 3b depicts the resulting secondary
structure analysis along with two representative AFM images of the
amyloid fibrils. It could be shown, that the small, worm-like amy-
loids of
b
-lg grown under high concentration (7.5% w/v) conditions,
exhibit a high content of
a
-helical/unordered structures. For the
long fibrils, formed at lower protein concentration (3% w/v), a
strong content of
b
-sheets could be detected. The fibril persistence
length (or bending rigidity) apparently decreases for an increasing
content of
a
-helical/unordered secondary structure. Whereas for
straight fibrils with a strong
b
-sheet content a persistence length of
3820 160 nm was found, the worm-like fibrils had a persistence
length of only 92 7nm[60]. While the influence of secondary
structure on the stiffness of amyloid fibrils could be unambiguously
demonstrated, the precise spatial distribution of secondary struc-
ture remains unclear. Structure sensitive methods with inherent
high spatial resolution like tip enhanced Raman scattering (TERS,
see also below) may in the future address the question how fibrils
can be built up with such remarkable differences in their secondary
structure composition, and yet be of the same thickness.
In vivo, the growth of amyloid fibrils, as well as the cytotoxic
effect of fibrils or their oligomeric precursors, is thought to take
place on the plasma membrane of cells [30,31]. The possibility of
vSFG to exclusively detect signals from model membranes like the
airewater interface of lipid monolayers provides the opportunity to
measure structural properties of amyloid fibrils under more bio-
logically relevant conditions than with conventional vibrational
spectroscopy techniques. In their pioneering work on amyloids
forming at lipid monolayer interfaces the Yan group employed
surface-selective SFG to monitor structural changes of islet amyloid
polypeptide during fibrillation [61,62]. Subsequent they reported
on the orientation of human IAPP amyloids, based on polarization
dependent SFG studies in combination with ab initio calculations
[63]. hIAPP is involved in the death of insulin producing islet cells of
Langerhans [29]. With SFG, the misfolding of hIAPP from an
a
-he-
lical or unordered form to a
b
-sheet rich amyloid structure was
kinetically resolved under the catalyzing action of negatively
charged phospholipids [61].
As mentioned in the background section, polyphenols like
()-epigallocatechin gallate (EGCG) are promising inhibitors of
amyloid formation in the context of various amyloid-related dis-
eases. Applying the experimental conditions employed by the Yan
group [61], we tested the inhibitory action of EGCG using the sur-
face specificity of SFG at the phospholipid interface. Fig. 4 sum-
marizes the experiments following the changes in secondary
structure of human IAPP in the amide I spectral region. Without
inhibitor, a structural transition from an
a
-helical or unordered
secondary structure to a
b
-sheet rich structure was observed by a
shift of the amide I band from 1652 cm
1
e1672 cm
1
[64]. This
secondary structure transition was interpreted as the formation of
amyloid fibrils (see Fig. 4a), as verified by AFM measurements on
the same samples transferred to a solid substrate. In the presence of
the inhibitor EGCG the structural transition is less pronounced, but
still obvious [64]. This observation is in strong contrast with the
situation in bulk. A standard biophysical technique for detecting
amyloids in solution is the Thioflavin T (ThT) fluorescence assay.
Upon binding of Thioflavin T to
b
-sheet rich amyloids, an enhanced
and red-shifted fluorescence is observed, as shown in Fig. 4c for the
Fig. 3. a. SFG spectra of
b
-lactoglobulin amyloids on mica in the amide I spectral region. Amyloid fibrils were grown for different monomer concentrations, as indicated in % w/v. The
spectra allow for a secondary structure analysis of the samples by fitting the vibrational SFG band with two components, corresponding to the contribution of
a
-helices or un-
ordered structures (around 1652 cm
1
) and
b
-sheets (around 1629 cm
1
). b. Quantitative analysis of the SFG spectra shows, that amyloid fibrils grown under higher concentration
conditions (6 and 7.5% w/v) exhibit higher
a
-helical or unordered structural content, and a less pronounced
b
-sheet structural content. AFM imaging of the
b
-lactoglobulin amyloid
samples on mica shows a morphology change from long, straight fibrils at low concentration to worm-like ones at higher concentrations (the white bar corresponds to a length of
100 nm). This transition is evidently accompanied by a change in secondary structure. Figures are based with permission on [60]. Copyright 2011 American Chemical Society (Minor
corrections of the peak fractions in b. were performed, which do not affect the general trend of the original figure).
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formation of IAPP amyloid fibrils in bulk solution (see trace marked
by blue ). In the presence of the inhibitor EGCG, no increase in
fluorescence could be detected (red þ). The combined experiments
at the interface and in bulk solution lead to the conclusion, that
whereas EGCG is a potent inhibitor in bulk, its inhibitive effect at
the membrane interface is strongly reduced. The membrane
interface represents the biologically relevant location of the cyto-
toxicity of hIAPP. Even more pronounced is the difference between
bulk and lipid interface for the disaggregation of pre-formed am-
yloids by EGCG. The ability of EGCG to dissolve preformed aggre-
gates in bulk solution could be verified again by a ThT assay as
depicted in Fig. 5a (blue ). The fluorescence rapidly decreases after
addition of the inhibitor, indicating a fast disaggregation. SFG is
able to monitor the effect of EGCG exclusively at the lipid interface
(red
D
). Even after incubation times up to 30 h (Fig. 5a) or after
addition of a 1000-fold molar excess of EGCG compared to the
hIAPP concentration (Fig. 5b), the
b
-sheet content of the sample
stays constant. AFM confirmed the presence of fibrils after treat-
ment with EGCG (see inset in Fig. 5b). In conclusion, at the lipid
interface the inhibitor EGCG is not able to dissolve amyloid fibrils at
all. The molecular origin of these findings is the subject of ongoing
research, but the results clearly highlight the importance of struc-
tural information gained selectively at interfaces.
vSFG offers many possibilities for future studies that can address
more complex, biologically relevant problems. For instance, the
inherent fast time-resolution of vSFG may be exploited to reveal
short lived oligomeric states during amyloid formation or disag-
gregation. While applications of whole cells in pharmaceutical
studies of amyloids are well documented [46,65], recently vSFG was
applied on fibrillar proteins of the extracellular matrix of living cells
as well. In future experiments, the complexity of in vivo tests is
therefore not beyond the means of vSFG [66].
3.2. Tip-enhanced Raman scattering (TERS)
A combination of the excellent spatial resolution of AFM and the
structural sensitivity of vibrational spectroscopy in a single setup is
realized by tip enhanced Raman spectroscopy (TERS). TERS is
becoming an increasingly off-the-shelve tool for label-free struc-
tural investigations of nanoscale phenomena at surfaces. Recent
results have demonstrated a lateral resolution down to the sub-
Fig. 4. a. Fibrillation of human islet amyloid polypeptide (hIAPP) followed by SFG at a phospholipid monolayer-covered water/air interface. At early times, the amide I band is
centered at 1652 cm
1
, corresponding to a mainly
a
-helical or unordered secondary structure. For later times a shift of the amide I band to 1672 cm
1
is observed, denoting a
b
-sheet
rich structure, characteristic for amyloids. b. hIAPP fibrillation followed in the presence of the inhibitor EGCG. The formation of
b
-sheets is less pronounced, but still evident. Fitting
of three bands (as exemplified for the earliest time by the broken red lines in a and b) to the SFG data allows a quantification of the
b
-sheet contribution to the spectrum. c. A
comparison of the
b
-sheet spectral contribution, revealed by SFG for the lipid interface situation and the fluorescence intensity of a Thioflavin T assay for bulk solutions. Both
situations are plotted in the absence (blue Bfor SFG and blue for ThT assay) as well as in the presence of the inhibitor EGCG (red
D
for SFG and red þfor ThT assay). The effect of
the inhibitor EGCG is strongly diminished at the interface, compared to the bulk situation. Adapted with permission from Ref. [64]. Copyright 2012 American Chemical Society.
Fig. 5. a. Disaggregation of preformed hIAPP fibrils, followed by the amount of
b
-sheet secondary structure using SFG at the phospholipid interface (red
D
) and the fluorescence
intensity of a ThTassay in bulk solution (blue ). At time zero, EGCG is added, which leads to a rapid fibril disaggregation in bulk, whereas no disaggregation takes place at the lipid
interface. b. Even a 1000 fold molar excess (1000
m
M) EGCG does not lead to a significant decrease in the
b
-sheet content. The presence of fibrils is verified by AFM imaging
(see inset). Based on [64] with permission. Copyright 2012 American Chemical Society.
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nanometer range. Clearly, this renders the method a valuable tool
for the direct investigation of single polymer strands.
One of the key components of a TERS setup is a plasmonic tip,
like a sharp edge of gold or an isolated gold or silver nanoparticle.
The external, monochromatic electromagnetic laser field oscillates
at a given frequency, which drives the electrons in the plasmonic
structure to perform a periodic motion. The resulting oscillating
dipole may increase the strength of the electromagnetic field in the
immediate vicinity of the plasmonic structure by many orders of
magnitude. Raman scattering can take place in molecules under the
plasmonic structure. Because both the incident and scattered
Raman fields are amplified by the plasmonic effect, the Raman field
scales linearly with the incident field, and because the detected
Raman intensity scales with square of the Raman field, the Raman
intensity enhancement scales with the fourth power of the local
field enhancement. As a result, extremely low concentrations down
to single molecules can be investigated. The size of the nano-
particles determines the spatial extent of the field-enhancing re-
gion and as a rule of thumb the lateral resolution in TERS is about
half of the radius of the nanoparticle. In addition further
enhancement mechanisms due to interactions between the metal
particle and the investigated molecule can occur (so called chem-
ical enhancement). Interestingly the depth resolution is even
higher than the lateral resolution. According to simulations the
electromagnetic field decays very fast and the z-resolution is in the
range of 1e2 nm. Also here, a direct interaction also increases this
depth resolution.
These properties are ideally suited to investigate amyloids with
a thickness of a few nm and length in the micrometer scale. The
correlation of a topographic image of a fibril with the spectral map
could possibly reveal the surface structure of complete fibrils. The
polymorphism of single amyloid fibrils gets therefore directly
accessible.
An inherent characteristic of TERS is the ability to analyze the
topographic and chemical structure of a specimen in one experi-
ment. Fig. 6 shows the results of such an experiment on a single
segregated hIAPP fibril. In Fig. 6a selected TERS spectra show the
typical Raman signals of a protein. The detection sites on the fibril
are marked in the topography image in Fig. 6b.
The spectral range in Raman spectroscopy that gives infor-
mation on the secondary structure of a specimen is the amide I
region between 1640 and 1680 cm
1
. The bands highlighted in red
(1660e1680 cm
1
)inFig. 6a are characteristic for
b
-sheet struc-
tures whereas the bands highlighted in green between 1640 and
1655 cm
1
are marker bands for
a
-helix structures. The detection of
both conformations on hIAPP fibrils indicates that the surface is
composed of a heterogenous secondary structure. This is different
from the core structure, which is known to be pure
b
-sheet. Such
observations have been made previously on insulin [67] fibrils and
give rise to the assumption that such heterogeneity is a common
feature of amyloid fibrils. The role of such conformation mixtures is
still under investigation.
The diversity of the spectra in Fig. 6a recorded on positions at
nanometers distance, demonstrates the ability of TERS to differ-
entiate protein structures on the nanometer scale. Besides a char-
acterization of secondary structures on fibrils this specialized
Raman spectroscopic technique provides information about the
amino acid distribution on the fibril, also with nanometer resolu-
tion [57]. It is evident that this approach can be of great assistance
for understanding the fibrillation process trigger and/or mecha-
nism on the nanometer scale.
In addition to having the ability to address amyloid fibril poly-
morphism, TERS holds the possibility to elucidate the role of single
amino acids in determining fibril stability and mechanical proper-
ties. This is an important issue, as the side chains of amino acids
have been shown to contribute to the stability of the
b
-cross core of
amyloids, affecting the elastic modulus of fibrils [3].
3.3. Coherent two-dimensional infrared spectroscopy (2DIR)
2DIR has become a powerful tool for the structural investigation
of proteins. By addressing localized vibrational transitions using a
specific sequence of infrared laser pulses, details of the molecular
response can be obtained, which can be related to structure and
structural evolution. The structural information contained in
respective linear spectra is essentially mapped onto two di-
mensions. The essence of 2DIR spectroscopy is that one specific
vibrational mode is selectively excited, and the effect of that exci-
tation is monitored on both that same mode, and on other vibra-
tional modes. From the study of the mode itself, the distribution of
vibrational frequencies can be obtained, as well as the timescale on
which these frequencies change, as a result of conformational
fluctuations, for instance. If the excitation has an effect on a
different mode, this means that the two molecular groups primarily
responsible for the modes are physically close to one another. The
strength and nature of the resulting coupling can be related to the
local structure, and structural rearrangement (basic details can be
obtained from the textbook [68]).
2DIR spectra can be recorded either in the frequency domain or
in the time domain, which both results in basically identical spectra
Fig. 6. a. Four raw TERS spectra obtained from a single IAPP fibril (generated at pH 7).
Spectra 1 and 2 were recorded at a relative lateral distance of 1.5 nm at the position
marked by the upper * in panel b. and show characteristic bands of
a
-helix secondary
structures (highlighted in green). Spectra 3 and 4 were recorded at a relative lateral
distance of 1.5 nm at the position marked by the lower * in panel b. and show in
contrast characteristic bands of
b
-sheet structures (highlighted in red). b. Typical AFM
topography of IAPP fibrils (generated at pH 7). TERS spectra were acquired on the top
fibril at two areas (marked with *), each with two positions separated by 1.5 nm,
respectively.
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[69]. Experiments in the frequency domain are realized by the
pump-probe technique. A scanning, spectrally narrow IR pump
pulse excites vibrational transitions and a spectrally broad probe
pulse identifies the exited vibrations after a fixed time from the
pump pulse. The 2D spectrum is constructed by plotting the
measured probe spectra for each applied pump frequency.
For 2DIR experiments in the time domain, also named echo
spectroscopy, three sequential IR pulses are applied to generate the
echo signal. The latter is recorded with a varying delay between the
two first (pump) pulses and a fixed delay between the second
pump and last (probe) pulses. Heterodyne detection is applied to
extract both amplitude and phase information. The construction of
a 2D spectrum involves a Fourier transformation: if the probe
spectra are plotted against a full set of the time delays between the
two pump pulses, a damped oscillation in the recorded intensity for
each probe frequency is yielded. This is analogous to multidimen-
sional FT-NMR correlation spectroscopy, where a free induction
decay (FID) is measured for a sequence of radio frequency pulses. By
means of Fourier transformation of the time-domain signal a pump
spectrum in the frequency domain is obtained for each measured
probe frequency.
A typical 2DIR spectrum contains valuable information, addi-
tionally to linear spectroscopy. Diagonal elements refer to peaks
near the diagonal of the 2D spectrum. They originate from excited
vibrational states, leading to higher order absorption (e.g. from the
first to the second excited state), resulting in positive peaks and
emission processes resulting in negative bands slightly shifted with
respect to the positive ones. Due to fast vibrational relaxation
processes occurring between pump and probe pulses, additional
negative bands may arise due to absorption of the probe beam from
vibrational ground states. The main characteristics of 2DIR spec-
troscopy are the cross-peaks, which arise from coupled vibrational
modes. Knowledge on vibrational coupling in terms of distance and
angular dependence can be transferred to structural information. In
complex systems like biomolecules often models based on molec-
ular dynamics simulations are implemented to extract structural
information from vibrational coupling [70]. 2D band shapes include
additional structural information and are influenced by environ-
ment effects such as hydrogen bonding or solvent effects [71].
Additionally to the structural sensitivity of 2DIR, transient 2DIR
permits probing of the kinetics of structural transitions with sub-
picosecond time resolution, for instance in case of chemical re-
actions [72,73].
A widely used application of 2DIR is the secondary structure
analysis of proteins. 2DIR is regarded as being more accurate than
1D FT-IR because of the increased information content achieved by
having information on both excitation and detection axes, and the
above-mentioned high time-resolution [74,75]. Isotopic labeling of
specific amino acids can be employed to reveal the site-specific,
local secondary structure of proteins [71]. Additionally, challenges
like heterogeneity within a protein, proteineprotein interactions or
ligand binding can be addressed [70]. Although secondary structure
analysis is quite accessible with 2DIR, investigations of the tertiary
structure of proteins are performed in combination with molecular
dynamics calculations to distinguish between different tertiary
structure types. A practical drawback of the technique is the
requirement of high sample concentrations, when compared to
traditional 1D FT-IR or monolayer sensitive vSFG.
A recent example of a detailed structural analysis of amyloids
from hIAPP was reported by Wang et al. [73]. The authors com-
bined experimental information from 2DIR spectroscopy with
calculations of 2DIR spectra and MD simulations of hIAPP, allowing
for conclusions on the structure of hIAPP fibrils beyond secondary
structure analysis. Based on the fact that the diagonal line widths
of the amide I peak of single amino acids differ for residues from a
b
-sheet and C-terminal or turn-structures, the authors deduced
the number of folds of
b
-structures within the peptide. Experi-
mentally, they employed seven samples with (diluted) site-specific
13
C¼
18
O labeled amino acids. It was therefore possible to test the
secondary structure of hIAPP specifically on seven distinct loca-
tions along the peptide chain. The diagonal amide I peaks of the
labeled amino acids showed a pronounced down shift compared
to the unlabeled amino acids and could be investigated mostly
undisturbed by the unlabeled part of the protein. Plotting the line
widths of labeled amide I peaks against the residue number, Wang
et al. observed a characteristic “W-shape”pattern. This pattern
was assigned to the presence of two stable
b
-sheet regions con-
nected by a turn-structure within a single amyloid-forming hIAPP
monomer. The pattern could be compared to calculated 2DIR
spectra based on previously proposed structural models of hIAPP
amyloids relying on solid state NMR and electron microscopy
studies [76,77].
The Zanni group, involved as well in the above mentioned study,
reported on the residue-specific kinetics of hIAPP aggregation [78],
employing isotopic labeled amino acids on six different positions
within the peptide. Based on the time-resolved site-specific for-
mation of
b
-sheets it was possible to conclude on requirements for
a model of the hIAPP aggregation pathway. Such a pathway was
found to be more complex than a simple pathway requiring a
random-coil intermediate, which undergoes a concerted transition
to a fiber-like nucleus and subsequent elongation by the addition of
monomers [78].
2DIR contributed further to the elucidation of structural tran-
sitions during the formation of amyloids [78e80], as well as the
effect of inhibitors on amyloid formation [81]. Meng et al. reported
on the effect of an inhibitor based on a sulfated triphenyl methane
derivate on the formation of amyloids from hIAPP. 2DIR revealed an
influence of the inhibitor on the
b
-sheet content of the samples, as
shown in Fig. 7.InFig. 7a, the 2DIR spectrum of the pure hIAPP
fibrils shows two diagonal out-of-phase features (doublets). A
strong one at the pump frequency of 1619 cm
1
, corresponding to a
predominant
b
-sheet secondary structure [71], as well as a weaker,
broad feature at 1646 cm
1
, consistent with an unordered structure
[81]. In the presence of inhibitor, a strong decrease in the peak
intensities attributed to the
b
-sheet structure is evident (Fig. 7b). A
smaller effect is an accompanying frequency shift of 3 cm
1
to
higher frequencies compared with pure hIAPP samples, which the
authors attributed to a smaller size of the
b
-sheets.
The interaction of a peptide-based inhibitor of hIAPP fibril for-
mation was investigated by Middleton et al. [28]. The authors
applied isotopic labeling for a residue-specific 2DIR analysis of the
inhibitory effect of rat IAPP (rIAPP) on hIAPP fibrillation. For this
purpose, they incubated the inhibitor and the amyloid peptide for a
short (8 h) and a long time (24 h) after mixing. rIAPP was known
not to form fibrils under any conditions, but represents a moderate
inhibitor of hIAPP fibril formation (for in vitro tests a strong inhi-
bition of fibril growth requires approximately a tenfold molar
excess of rIAPP [82]). A first finding was the identification of the
inhibitor binding side by the fact that the N-terminal
b
-sheet of
hIAPP is not formed within 8 h in the presence of rIAPP (Fig. 8a).
This result explained the structural basis of the slower amyloid
formation in the presence of the inhibitor. After long-time incu-
bation of inhibitor and hIAPP, fibril formation was not inhibited.
Surprisingly it was found, that rIAPP and hIAPP form mixed amy-
loidogenic parallel
b
-sheets 24 h after mixing (Fig. 8b). The struc-
tural sensitive 2DIR technique could reveal therefore a potential
additional toxicity of an agent, which was previously thought to
combat amyloid formation. Structure sensitive 2DIR proved
therefore as a very fruitful technique for the investigation of amy-
loid formation and inhibitor interaction, in particular because
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standard fluorescence techniques (Thioflavin T assay) suggested
that the system already equilibrated after 8 h [28], contrary to 2DIR.
More complex experiments like 3DIR approaches or additional
pulse sequences [68] may in future further strengthen the appli-
cation of multidimensional IR correlation spectroscopy to struc-
tural characterization of biopolymers.
4. Mesoscopic properties of single amyloid fibrils
Proteins and peptides form amyloid fibrils by self-assembling
into protofibrils, which have a
b
-sheet core. Usually, between 2
and 4 protofibrils subsequently twist together to form a mature
fibril [83e85]. Amyloid fibrils tend to be highly polymorphic,
varying in length and in the number of protofibrils. The
b
-sheet
core makes amyloid fibrils rigid with a Young’s modulus similar to
that of silk and strong with an ultimate tensile strength comparable
to that of steel [2]. Because of their outstanding mechanical prop-
erties, amyloid fibrils are promising structures for applications in
biomaterials and food technology. However, the mechanical prop-
erties of amyloids are still not fully understood. There are several
theoretical models which assign the large rigidity and strength of
amyloid fibrils to their
b
-sheet core [3,86]. However, amyloid fibrils
are polymorphic, and vibrational spectroscopy data indicate that
the
b
-sheet content can be much less than 100%, as shown in Fig. 3.
At least in part, this is a consequence of the presence of side-chains,
whose effect on the mechanical properties of fibrils is unclear. In
addition, amyloid fibrils may have imperfect
b
-sheet cores with
interruptions along the long axis. Here we will review recent bio-
physical studies of the mechanical properties of single fibrils, and
indicate some promising techniques that can be used in future to
better understand the mechanics of amyloid fibrils and their origin.
4.1. Bending rigidity and shear modulus
A key parameter characterizing the conformation of a
biopolymer is the bending rigidity. The bending rigidity is often
expressed by the length scale beyond which the fibril shows sig-
nificant curvature due to thermal forces, quantified by the persis-
tence length P[87,88]. The bending rigidity can be measured by a
variety of methods, which are either based on active deformation of
the fibrils, or on analysis of the spontaneous, thermal bending
fluctuations of the fibrils.
Active deformation of fibrils can be achieved by AFM, as
demonstrated in a recent study on insulin amyloid fibrils composed
of two protofibrils [2,3]. Essentially, the experiment was a micro-
scopic equivalent of a traditional three-point bending test: fibrils
were deposited on a silicon substrate with nanoscale grooves. With
an AFM probe, a controlled load was applied on fibrils suspended
over a groove, while monitoring the deflection of the cantilever that
acts as the force sensor. The Young’s modulus was determined from
the linear (small-force) part of the deflection curve, giving an
average Young’s modulus of E¼3.3 0.4 GPa and shear modulus of
G¼0.28 0.2 GPa. This Young’s modulus corresponds to a
persistence length of 42 30
m
m, which is much larger than the
typical fibril contour length of 3e6
m
m. An advantage of this
technique is that the measurements are performed on fibrils in
their natural, hydrated state. However, it is only possible to mea-
sure on the length scale of the tip radius, which is small compared
to the fibrils. Also, the experiment is technically very challenging
because of the small diameter of the amyloid fibrils [83].
Technically, it is far easier to measure the bending rigidity based
on the spontaneous, thermal fluctuations of fibrils. The basic idea is
to measure the shape of fluctuating fibrils, either taking snapshots
of a large ensemble of fibrils at a given moment in time, or taking
time-lapse movies of a single fibril. Until now, most studies of
amyloid fibrils used the first method, analyzing the shape of a large
ensemble of fibrils immobilized on a surface. Usually, amyloid fi-
brils are imaged by AFM [60,89e91], which requires fibril deposi-
tion on a mica or glass surface and drying. Alternatively, cryo-
transmission electron microscopy (cryo-TEM) can be used, which
has the benefit that fibrils are preserved (snap-frozen) in a hydrated
state [92]. The persistence length Pcan be calculated by measuring
Fig. 8. Model structures of the co-aggregated hIAPP (grey) and rIAPP (pink) a. 8 h and
b. 24 h after mixing of the peptides. One site-specific isotopic labeling side of hIAPP is
depicted as blue spheres. In a., rIAPP prevents initially the N-terminal formation of
b
-sheets of hIAPP. In b., mixed
b
-sheets from hIAPP and rIAPP are formed. Reused with
permission from Ref. [28]. Copyright 2012 Macmillan Publishers.
Fig. 7. 2DIR spectra in the amide I spectral region of hIAPP after formation of amyloids
in the (a) absence or (b) presence of an inhibitor based on a sulfonated triphenylmethyl
derivate. The same growth conditions were applied. Reprinted with permission from
Ref. [81].
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for each fibril the contour length, C, and the end-to-end distance, E
(Fig. 9). According to the worm-like chain model, the mean-squared
end-to-end distance on a two-dimensional surface as a function of
Cdepends on Pas in the following equation:
E
2
2D
¼4PC12P=C1e
C=2P
:
This equation assumes that the fibrils interact weakly with the
surface and can relax to a two-dimensional equilibrium confor-
mation [87,89,91]. However, if the interaction between the fibril
and the surface is much stronger than the thermal energy, the fi-
brils will be trapped by the surface, leading to more condensed
fibril conformations. In this case, the mean-squared end-to-end
distance of the fibril amounts to [89]:
E
2
3D
¼4=3PC1P=C1e
C=P
:
An alternative method of determining Pis by analyzing the
correlation of bond angles along the contour:
P¼hli=ð1hcos
4
iÞ;
where <l>is the average segment length and
4
is the angle be-
tween segments (Fig. 9)[93].
One of the best-studied types of amyloid fibrils are those formed
from the model protein
b
-lactoglobulin (
b
-lg). Depending on the
self-assembly conditions, these fibrils have widely varying persis-
tence lengths and mostly fall into one of two classes: straight fibrils
or worm-like fibrils (Fig. 10a, b and Fig. 3b). Straight fibrils generally
have a persistence length that is comparable to the contour length,
ranging from about 600 nm to several
m
m[37,60,90,91,93e96].
Worm-like fibrils are much more flexible, with a persistence length
of only 10e90 nm (Table 1)[37,60,89,91,93,94,96]. A third type of
morphology, rod-like fibrils with a persistence length of 135 nm,
was formed from
b
-2-microglobulin (Fig. 10c) [91]. It has to be
noted that amyloid fibrils are polymorphic; even under a given set
of conditions, fibrils have different persistence lengths. The
numbers given in Table 1 are average values.
In contrast to nanomechanical manipulation assays, the fluctu-
ation analysis approach gives a global measure of the persistence
length of a fibril, because fluctuations are evaluated on a larger
length scale. However, fibril imaging by AFM requires deposition on
a surface and drying, which can potentially lead to artifacts.
One way to overcome this problem is to measure the shape of
freely fluctuating fibrils in solution by fluorescence microscopy
(Fig. 10d) [97,98]. Because of the low resolution of optical micro-
scopes in the z-direction, fibrils are typically confined in a quasi-2D
geometry by sandwiching them between two glass cover slips.
Rather than extracting the persistence length from the static shape
of an ensemble of fibrils, the persistence length is now calculated by
tracking the fibril shape fluctuations over time. These experiments
have been performed for yeast prion fibrils labeled with a
fluorescent dye [98]. The persistence length was found to be
3.6 1.1 or 7. 0 2.4
m
m, depending on the assembly conditions. In
addition to the bending rigidity, also the bending dynamics can be
determined from time-lapse movies, as demonstrated for actin
filaments and microtubules [88].
A second way to overcome problems associated with drying or
surface immobilization is to perform light scattering of dilute fibril
suspensions, again using the worm-like chain model to interpret
the data. The persistence length of
b
-lg amyloid fibrils formed at
varying ionic strengths was determined by a combination of light
scattering (LS) and small-angle neutron scattering (SANS) [94]. The
persistence length decreased with increasing ionic strength from
600 to 38 nm. These values are in reasonable agreement with the
persistence lengths obtained with imaging techniques (Table 1).
The persistence length of
b
-lg fibrils also has been estimated
using an adjusted random contact model, based on measurements of
the storage (or elastic) modulus G
0
of fibril gels by rheology [37]. The
critical percolation concentration was determined by measuring G
0
as a function of protein concentration. It was assumed that for a
fibrillar system the percolation mass fraction is described by the
volume of the fibril, the number of contacts per rod and the excluded
volume of charged semiflexible fibrils.The volume of the fibril can be
determined by the effective diameter, contour length and persis-
tence length. The estimated persistence length was 1.6 0.4
m
m for
b
-lg fibrils formed at pH ¼2 and 80
C(Table 1, 7th entry).
4.2. Ultimate tensile strength and fracture behavior
The ultimate strength of single fibrils has been measured by
nanomechanical manipulation with AFM. For insulin fibrils, the
ultimate strength was measured by actively bending fibrils sus-
pended over a groove with an AFM tip. The mean ultimate strength
for fibrils composed of two protofibrils was 0.6 0.4 GPa. Strik-
ingly, this is in the same order of magnitude as the strength of steel
(0.6e1.8 GPa) and silk (1.0e1.5 GPa) [2,3]. The corresponding
breakage force was estimated to be in the range of 300e500 pN [2].
Microscopic insight into the molecular mechanisms that deter-
mine the strength of amyloid fibrils can be obtained by force
spectroscopy, where peptide strands are pulled from fibrils
immobilized on a surface with a small AFM tip. Such experiments
were reported for fibrils formed from A
b
(1e40) or A
b
(25e35)
peptides [99]. Strands of more than 100 nm in length could be
pulled out of the fibrils and stretched. The force-extension behavior
that was observed was fitted with a worm-like chain model, from
which a persistence length of about 0.4 nm was calculated. Based
on these results, the authors concluded that the strands were
b
-
sheets that were unzipped from the fibrils. The unzipping process
was fully reversible. Fibrils formed from A
b
(1e42) peptide showed
a lower unzipping force (w23 pN) than fibrils formed from A
b
(1e
40) peptide (w33 pN) [99,100]. Unzipping experiments were also
attempted on amyloids formed from Als cell adhesion proteins of a
fungal pathogen, but in this case unzipping of mature fibrils was
not possible. However, zipper interactions were detected between
monolayers of Als proteins and an Als amyloid sequence attached to
an AFM tip. The characteristic force signatures corresponded to the
mechanical unzipping of
b
-sheet interactions formed between
parallel Als proteins, and was about 30 pN [101].
The weak point of the above mentioned zipping experiments is
that the measurements were based on nonspecific binding be-
tween tip and fibril. Therefore, the variability between individual
measurements likely results from random and multiple attachment
sites of the protein to the tip. The nanomechanical properties of
human prion protein amyloid (huPrP90-231) were investigated
using specific binding between the AFM tip and the fibril [102]. The
protein was functionalized by replacing one amino acid for a
Fig. 9. Schematic depiction of the end-to-end length E, the segment length l, and the
angle between segments 4, for a general semiflexible filament.
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cysteine, which is known to bind covalently with a gold-coated
AFM tip. Again, force-extension curves showed elastic stretching
for small retractions, characteristic of entropic stretching of a
disordered peptide chain between the
b
-sheet core and the cova-
lent attachment to the tip. At larger retractions of the tip, rupture
occurred, which likely reflects the mechanical extraction of the
prion protein from the core of the amyloid fibril. The force at
which rupture took place, was 115 5 pN at a tip retraction rate of
9.4 nN/s, and was dependent on the loading rate.
Even though amyloid fibrils are generally regarded mechanically
strong, they do break under the influence of thermal forces. In fact,
spontaneous fracture is thought to be a key feature of the kinetics of
fibril growth, since fragmentation increases the number of free
ends, thus enhancing the rate of fibril growth [2,103]. It has also
been shown that amyloid fibrils break easily by elongational flow
[104]. Whey protein isolate (WPI) amyloid fibrils already fracture at
an elongational flow rate of only 8 s
1
, which is much lower than
the flow rate where DNA strands break, which is close to 10,000 s
1
.
The fracture force estimated from these observations are 0.1 pN
from the extensional flow experiment and 4 pN from the stability of
fibrils against thermal force [104]. It is still unclear how these low
fracture forces may be reconciled with the much higher fracture
forces of 300e500 pN estimated from active nanomanipulation
experiments of insulin fibrils [2]. It will be interesting to combine
mechanical measurements with vibrational spectroscopy, to link
the fracture strength to the underlying molecular structure, which
may potentially depend on the peptide used as well as on the as-
sembly conditions.
4.3. Extensibility
It was recently shown that the fundamental structural unit of
several kinds of amyloid fibrils including A
b
is the
b
-helix structure
[105e107]. The
b
-helix is a protein motif formed by the association
of parallel
b
-strands in a helical pattern with either two or three
faces (Fig. 11)[84]. The mechanical properties of this structure
under tension and compression were studied with molecular dy-
namics (MD) simulations. The calculated maximum tensile force
was 522 pN, while the maximum compressive force was much
higher, namely 3150 pN. The simulations revealed that the
b
-helix
structure is extremely extensible and can sustain tensile strains up
to 800% without rupture of the covalently bonded protein back-
bone [84]. The model that was used only accounts for the core
structure of certain amyloids, not including effects of side chains. It
will be interesting to test by experiments the prediction that am-
yloid fibrils should be highly extensible.
4.4. Outlook
Although there is quite a large number of studies of the bending
rigidity of single amyloid fibrils, most of these rely on AFM imaging
Fig. 10. AFM images of long, straight a. and worm-like b.
b
-lg amyloid fibrils. Scale bars are 500 nm. Reprinted with permission from VandenAkker et al. [60], J. Am. Chem. Soc. 2011,
133. c. Rod-like
b
-2-microglobulin amyloid fibrils. Image size is 1 1
m
m. Reprinted with permission from Gosal et al. [91], J. Mol. Biol. 2005, 351. d. Fluorescence microscopy image
of single yeast prion amyloid fibrils labeled with Thioflavin T. Scale bar is 2
m
m. Reprinted with permission from Castro et al. [98], Biophys. J. 2011, 101.
Table 1
Persistence lengths of straight and worm-like
b
-lg amyloid fibrils, measured using
different techniques.
Reference Morphology Technique Persistence length
VandenAkker [60], 2011 Straight AFM 3820 nm 160
Adamcik [90], 2010 Straight AFM 968e3240 nm
Jordens [96], 2011 Straight AFM 2370 nm
Mudgal [93], 2009 Straight TEM 788 nm
Veerman [95], 2002 Straight TEM 1000 nm
Aymard [94], 1999 Straight LS, SANS 600 nm
Sagis [37], 2004 Straight Rheology 1600 nm 400
VandenAkker [60], 2011 Worm-like AFM 92 nm 7
Jordens [96], 2011 Worm-like AFM 29 nm
Mudgal [93], 2009 Worm-like TEM 36 nm 12
Aymard [94], 1999 Worm-like LS, SANS 38 nm
M. Schleeger et al. / Polymer xxx (2013) 1e16 11
Please cite this article in press as: Schleeger M, et al., Amyloids: From molecular structure to mechanical properties, Polymer (2013), http://
dx.doi.org/10.1016/j.polymer.2013.02.029
of fibrils deposited on a substrate and dried. It is unclear how surface
immobilization and drying may affect the properties of the fibrils.
Several techniques can be used to circumvent these experimental
limitations. A promising method is to measure the thermal fluctu-
ations of freely fluctuating amyloid fibrils with fluorescence mi-
croscopy, as was demonstrated for yeast prion amyloids [98].
Several studies have been reported of active mechanical manipu-
lation of amyloid fibrils. In one study, amyloid fibrils were bent by an
AFM tip, and in a few studies
b
-sheet segments were unzipped from
fibrils by AFM. A promising technique to measure both the bending
and stretch rigidity of amyloid fibrils is by a dual optical tweezers
assay. As demonstrated in a recent study of microtubules and actin
filaments, micrometer-sized beads can be attached to the two ends
of a biopolymer and used as handles to stretch the biopolymer by
two laser tweezers [108]. From the force-extension behavior, the
persistence length of single, hydrated fibers could be determined
based on well-controlled active experiments. Optical tweezers have
also been used to measure the elastic moduli of individual fibers in
networks of the blood clotting protein fibrin [109]. Micron-sized
beads were attached to fibers after clot formation and trapped
with optical tweezers. The bending and stretch rigidity of individual
fibers was measured by applying an oscillatory displacement to the
bead either orthogonally or tangentially to the fiber.
5. Microscopic properties of amyloid networks
For applications of amyloid fibrils in food products, tissue en-
gineering, and materials sciences, the mechanical properties of
networks are relevant. Network mechanics is determined by a
combination of fibril mechanics and the spatial organization of fi-
brils and their interactions. The spatial organization of amyloid
fibril networks depends on fibril rigidity: when the fibrils are long,
thin, and rigid, they can form liquid crystalline phases or gels
already at low concentrations [110]. The interactions between fi-
brils are not well-studied, but are thought to be highly dependent
on the side chains on the surface of the fibrils, which can for
instance confer a pH-dependent electrostatic charge to the fibrils
[83]. Bulk rheology has been used to probe the mechanical prop-
erties of networks of amyloid fibrils [93,111,112]. The networks
generally form weak viscoelastic gels. However, quantitative com-
parisons between rheology measurements and theory are still
lacking, since the fibril morphology was not well-defined.
5.1. Rheological properties
Rheological properties relate to the flow properties of materials
in response to an applied deformation. The most commonly used
type of deformation is a shear deformation, where a material be-
tween two parallel plates is deformed by moving one of the plates
while the other is kept stationary. Typically, one applies either a
steady shear (constant shear rate) or an oscillatory shear of
controlled strain amplitude and frequency, and measures the stress
response. When the material is a Newtonian fluid such as water, the
stress in a steady shear measurement is proportional to the applied
shear rate with a constant of proportionality that is given by the
steady-shear viscosity. The viscosity is independent of the shear
rate. In contrast, polymeric materials are usually viscoelastic and
non-Newtonian, with a viscosity that does depend on shear rate.
Typically, the viscosity is constant at low strain rates, but decreases
when the strain rate is raised, a response that is known as shear
thinning. When the polymer material is elastic, a more suitable
measurement is an oscillatory shear measurement, which mea-
sures the complex shear modulus G*, which is the constant of
proportionality between the stress and the strain. G* is a complex
quantity with a storage modulus G
0
that reflects the elastic stress
response that is in-phase with the applied strain, and a loss
modulus G
00
that reflects the viscous stress response that is out-of-
phase with the applied strain [110]. Until now, oscillatory mea-
surements of amyloid networks have primarily focused on
resolving the gelation time and critical percolation concentration of
fibrils formed from food-related proteins including
b
-lg, bovine
serum albumin (BSA) and ovalbumin at pH ¼2[37,112e114]. Steady
shear measurements were used to measure the shear-rate depen-
dence of the viscosity of suspensions of whey protein fibrils with
lengths of several
m
m[111]. In all cases the suspensions were shear-
thinning [111,113].
Oscillatory measurements have also been used to measure the
influence of pH and ionic strength on gel strength. At pH ¼3.35, the
whey protein
b
-lg forms worm-like fibrils with a persistence length
of 35 nm and a diameter of 5 nm [93]. The viscosity of these fibrillar
networks was observed to increase with protein concentration. In
the presence of NaCl, fibril networks remained predominantly
elastic (with G
0
at least 10-fold higher than G
00
) up to ionic strengths
of 100 mM, but became weaker and eventually fluid-like above
200 mM NaCl (Fig. 12)[115]. BaCl
2
and MgCl
2
caused a significantly
Fig. 11. Structures of a. twofold and b. threefold symmetric assemblies of A
b
(1e40) amyloid fibrils. Reprinted with permission from Xu et al. [86], Biophys. J. 2010, 98.
M. Schleeger et al. / Polymer xxx (2013) 1e1612
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increased final viscosity of whey protein (WPI) gels compared to
gels formed without salt [113]. Also monovalent salts increased the
viscosity slightly compared to control fibrils formed without salt.
All WPI fibrils formed in the presence of salt, showed a worm-like
morphology, while fibrils formed without salt are generally long
and straight. Frequency sweep and strain sweep experiments
demonstrated that G
0
was relatively insensitive to added salt, but
the loss modulus G
00
was slightly higher with divalent salts than for
samples without or with monovalent salts [113]. The viscosity de-
pends on different parameters, including the volume fraction of
fibrils, the fibril morphology and the interactions between fibrils.
Because the effect of mono- and multivalent salts on these pa-
rameters was not determined, the mechanism behind the increase
in viscosity is still not understood.
There are few rheological studies of amyloids other than whey
proteins-related fibrils. Weak, solid-like behavior was also reported
for hydrogels formed from amyloids of the Alzheimer related A
b
peptide, A
b
(16e20) formed at high concentrations (3% w/v) [112].
An alternative method to measure the rheology of soft poly-
meric materials is by microrheology (MR), which is a collection of
techniques used to determine the local properties of a material
from the motion of embedded, small tracer particles. The most
popular MR technique is video particle tracking, where the ther-
mally induced motions of the tracer particles are observed by op-
tical microscopy [116]. Particle tracking is a convenient method to
obtain a spatial map of variations in the viscoelastic properties of
heterogenous samples [117]. An advantage of MR over bulk
rheology is the possibility to use small sample volumes (down to
5
m
L) and the possibility to determine the rheology over a broad
frequency range. Particle tracking MR has been used to study the
solegel transition in amyloid networks. Gels of
b
-lg fibrils were
prepared at pH ¼7 and room temperature by adding alcohol [116].
Under these conditions, wormlike fibrils are formed [118]. Latex
particles with a diameter of 0.5
m
m were used to observe the time
evolution of network formation for
b
-lg concentrations at and
above the critical gelation concentration of 4% w/v. In time, a shift
from purely viscous to viscoelastic behavior was observed, indica-
tive of fibril formation. Eventually, a solid gel was formed. Based on
the mean squared displacement (MSD) of the probe particles, the
elastic and loss moduli over a large frequency range could be
Fig. 12. Rheological characterization of 2% w/v
b
-lg protein fibrils at varying ionic strengths: A. 50 mM, B.100 mM, and C. 200 mM NaCl. D. Storage modulus determined at different
ionic strengths by frequency sweeps. E. Structural recovery behavior of the fibril gel prepared at the ionic strength of 50 mM as a function of time after destruction of the gel by a
100% oscillatory shear strain. Reprinted with permission from Ref. [115].
M. Schleeger et al. / Polymer xxx (2013) 1e16 13
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dx.doi.org/10.1016/j.polymer.2013.02.029
calculated (Fig. 13). Similar experiments were also performed for
b
-lg amyloid fibrils formed by decreasing the pH to 2 and heating to
80
C. Under these conditions, the fibrils that are formed are long
and straight, and the critical concentration for gel formation
was less than 3% w/v. The gels that were formed after about
100 min showed behavior typical of weak gels, with a plateau in G
0
that became more pronounced at longer incubation times up to
200 min [119].
5.2. Outlook
Particle tracking microrheology has a frequency range that is
limited by the camera acquisition rate and also by the inherent
localization error of ca. 20e50 nm in the particle position. These
limitations can be overcome by using laser tweezers combined
with quadrant photodiodes for sub-nanometer localization of
probe particles embedded in a network. By using weak laser beams,
the thermal fluctuations of the particles can be detected by laser-
interferometry. As shown for actin networks and worm-like mi-
celles, this method affords a wide frequency window of 0.1e
100 kHz, giving at the same time access to network properties
(below w1 kHz) and single fibril properties (above w10 kHz)
[120,121]. Optical tweezers can also be used for active MR, where a
probe particle is actively moved by a laser trap at controlled fre-
quency and amplitude. This method in principle allows measure-
ment of non-linear viscoelastic properties.
6. Conclusions/perspective
We have illustrated here how insights into amyloid chemistry
and physics on different length scales ranging from the molecular
to the macroscopic level can contribute to an improved under-
standing of amyloids and amyloid networks. The overarching goal
of these efforts is to obtain a comprehensive understanding of the
relation between secondary structure, amino acid distribution and
fibril assembly on the one hand and morphology and mechanical
properties of amyloids and networks thereof on the other hand.
Network mechanics are necessarily a function of fibril mechanics,
the architecture of networks, and interactions between fibrils. Fi-
brils mechanics are determined by the assembly and (core-)
structure of fibrils, as well as interactions of single amino acids
within the fibrils. It is evidently challenging to bridge the length
scales from the molecular morphology, through single fibril me-
chanical properties, to the macroscopic rheological properties for
these highly complex biopolymers, but some of the examples
presented above demonstrate significant progress in this field. The
recent development and application of advanced vibrational
spectroscopic techniques to these complex biological systems show
promise: several examples exist where a combination of tech-
niques focusing on the properties of amyloids at different length
scales was used. For example, the combination of vSFG and AFM
allowed relating the persistence length of different assembly con-
ditions to the global secondary structure composition of amyloids.
The change in morphology and persistence length could be shown
to be accompanied by a change in the secondary structure content
of the amyloid samples. Sophisticated FT-IR and Raman spectros-
copy can provide a comprehensive understanding of the secondary
structure of amyloids [52,53,118], and may be combined with me-
chanical deformation in the same setup, as shown for other bio-
polymers [122]. The advent of several new vibrational spectroscopy
techniques and microrheology approaches offers the possibility to
interrogate macroscopic, mesoscopic and microscopic properties of
amyloid fibrils.
Acknowledgment
This work is supported by NanoNextNL, a micro and nano-
technology consortium of the Government of the Netherlands and
130 partners, as well as part of the Industrial Partnership Pro-
gramme (IPP) Bio(-Related) Materials (BRM) of the Stichting voor
Fundamenteel Onderzoek der Materie (FOM), which is financially
supported by the Nederlandse Organisatie voor Wetenschappelijk
Onderzoek (NWO). The IPP BRM is co-financed by the Top Institute
Food and Nutrition and the Dutch Polymer Institute.
References
[1] Vollrath F, Porter D. Polymer 2009;50(24):5623e32.
[2] Smith JF, Knowles TP, Dobson CM, Macphee CE, Welland ME. Proc Natl Acad
Sci U S A 2006;103(43):15806e11.
[3] Knowles TP, Fitzpatrick AW, Meehan S, Mott HR, Vendruscolo M, Dobson CM,
et al. Science 2007;318(5858):1900e3.
[4] Nyrkova IA, Semenov AN, Aggeli A, Bell M, Boden N, McLeish TCB. Eur Phys J
B 2000;17(3):499e513.
[5] Dinca V, Kasotakis E, Catherine J, Mourka A, Ranella A, Ovsianikov A, et al.
Nano Lett 2008;8(2):538e43.
[6] Scheibel T, Parthasarathy R, Sawicki G, Lin XM, Jaeger H, Lindquist SL. Proc
Natl Acad Sci U S A 2003;100(8):4527e32.
[7] Hamada D, Yanagihara I, Tsumoto K. Trends Biotechnol 2004;22(2):93e7.
[8] Maji SK, Schubert D, Rivier C, Lee S, Rivier JE, Riek R. PLoS Biol 2008;6(2):
240e51.
[9] Gelain F, Bottai D, Vescovi A, Zhang S. PLoS One 2006;1:e119.
[10] Hu KN, McGlinchey RP, Wickner RB, Tycko R. Biophys J 2011;101(9):2242e50.
[11] Berryman JT, Radford SE, Harris SA. Biophys J 2011;100(9):2234e42.
[12] Kodali R, Wetzel R. Curr Opin Struct Biol 2007;17(1):48e57.
[13] Sipe JD, Cohen AS. J Struct Biol 2000;130(2e3):88e98.
[14] Lin JC, Liu HL. Curr Drug Discov Technol 2006;3(2):145e53.
[15] Iconomidou VA, Hamodrakas SJ. Curr Protein Pept Sci 2008;9(3):291e309.
[16] Jahn TR, Makin OS, Morris KL, Marshall KE, Tian P, Sikorski P, et al. J Mol Biol
2010;395(4):717e27.
[17] Wiltzius JJ, Sievers SA, Sawaya MR, Cascio D, Popov D, Riekel C, et al. Protein
Sci 2008;17(9):1467e74.
[18] Wetzel R, Shivaprasad S, Williams AD. Biochemistry 2007;46(1):1e10.
[19] Knowles TP, Smith JF, Craig A, Dobson CM, Welland ME. Phys Rev Lett
2006;96(23):238301.
[20] Guo S, Akhremitchev BB. Biomacromolecules 2006;7(5):1630e6.
[21] Pastor MT, Esteras-Chopo A, Lopez de la Paz M. Curr Opin Struct Biol
2005;15(1):57e63.
[22] Hamley IW. Angew Chem Int Ed Engl 2007;46(43):8128e47.
Fig. 13. Elastic and loss moduli of pre-gel (lower two curves) and post-gel (upper two
curves) systems versus frequency, a and b are shift factors. For clarity, the postgel
curves are shifted upward by a factor of 10
2
, and only every fourth data point is plotted
for each curve. The pregel curve shows viscous behavior at low frequencies (a slope of
1 for G00), while at high frequencies the moduli approach critical behavior and the
power law exponent is 0.59, comparable to the MSD power law exponent at the gel
point. Reprinted with permission from Corrigan et al. [116] Langmuir, 2009, 25.
Copyright 2009 American Chemical Society.
M. Schleeger et al. / Polymer xxx (2013) 1e1614
Please cite this article in press as: Schleeger M, et al., Amyloids: From molecular structure to mechanical properties, Polymer (2013), http://
dx.doi.org/10.1016/j.polymer.2013.02.029
[23] Baldwin AJ, Bader R, Christodoulou J, MacPhee CE, Dobson CM, Barker PD.
J Am Chem Soc 2006;128(7):2162e3.
[24] Hamedi M, Herland A, Karlsson RH, Inganas O. Nano Lett 2008;8(6):1736e40.
[25] Cellmer T, Bratko D, Prausnitz JM, Blanch HW. Trends Biotechnol 2007;25(6):
254e61.
[26] Sinha S, Lieberburg I. Proc Natl Acad Sci U S A 1999;96(20):11049e53.
[27] Lansbury PT, Lashuel HA. Nature 2006;443(7113):774e9.
[28] Middleton CT, Marek P, Cao P, Chiu CC, Singh S, Woys AM, et al. Nat Chem
2012;4(5):355e60.
[29] Höppener JWM, Ahrén B, Lips CJM. N Engl J Med 2000;343(6):411e9.
[30] Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, et al.
Science 2003;300(5618):486e9.
[31] Engel MFM, Khemtémourian L, Kleijer CC, Meeldijk HJD, Jacobs J, Verkleij AJ,
et al. Proc Natl Acad Sci U S A 2008;105(16):6033e8.
[32] Butterfield SM, Lashuel HA. Angew Chem Int Ed 2010;49(33):5628e54.
[33] Kontopidis G, Holt C, Sawyer L. J Dairy Sci 2004;87(4):785e96.
[34] Gosal WS, Clark AH, Ross-Murphy SB. Biomacromolecules 2004;5(6):2420e9.
[35] Gosal WS, Clark AH, Ross-Murphy SB. Biomacromolecules 2004;5(6):2430e8.
[36] Arnaudov LN, de Vries R. Biomacromolecules 2006;7(12):3490e8.
[37] Sagis LM, Veerman C, van der Linden E. Langmuir 2004;20(3):924e7.
[38] Arnaudov LN, de Vries R. J Chem Phys 2007;126(14):145106.
[39] Padrick SB, Miranker AD. Biochemistry 2002;41(14):4694e703.
[40] Nanga RPR, Brender JR, Vivekanandan S, Ramamoorthy A. Biochim Biophys
Acta Biomembr 2011;1808(10):2337e42.
[41] Nielsen JT, Bjerring M, Jeppesen MD, Pedersen RO, Pedersen JM, Hein KL,
et al. Angew Chem Int Ed Engl 2009;48(12):2118e21.
[42] Kapurniotu A. Biopol Pept Sci 2001;60(6):438e59.
[43] Ono K, Yoshiike Y, Takashima A, Hasegawa K, Naiki H, Yamada M.
J Neurochem 2003;87(1):172e81.
[44] Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, et al. Nat
Struct Mol Biol 2008;15(6):558e66.
[45] Cheynier V. Am J Clin Nutr 2005;81(1 Suppl.):223Se9S.
[46] Bieschke J, Russ J, Friedrich RP, Ehrnhoefer DE, Wobst H, Neugebauer K, et al.
Proc Natl Acad Sci U S A 2010;107(17):7710e5.
[47] Fresco P, Borges F, Diniz C, Marques MP. Med Res Rev 2006;26(6):747e66.
[48] Manach C, Scalbert A, Morand C, Remesy C, Jimenez L. Am J Clin Nutr
2004;79(5):727e47.
[49] Shoval H, Lichtenberg D, Gazit E. Amyloid 2007;14(1):73e87.
[50] Azriel R, Gazit E. J Biol Chem 2001;276(36):34156e61.
[51] Necula M, Kayed R, Milton S, Glabe CG. J Biol Chem 2007;282(14):10311e24.
[52] Oboroceanu D, Wang LZ, Brodkorb A, Magner E, Auty MAE. J Agric Food
Chem 2010;58(6):3667e73.
[53] Oboroceanu D, Wang LZ, Kroes-Nijboer A, Brodkorb A, Venema P, Magner E,
et al. Int Dairy J 2011;21(10):823e30.
[54] Arnolds H, Bonn M. Surf Sci Rep 2010;65(2):45e66.
[55] Pettinger B, Schambach P, Villagomez CJ, Scott N. Annu Rev Phys Chem
2012;63:379e99.
[56] Bailo E, Deckert V. Chem Soc Rev 2008;37(5):921e30.
[57] Deckert-Gaudig T, Kammer E, Deckert V. J Biophotonics 2012;5(3):215e9.
[58] Bonn M, Bakker HJ, Ghosh A, Yamamoto S, Sovago M, Campen RK. J Am Chem
Soc 2010;132(42):14971e8.
[59] Sovago M, Vartiainen E, Bonn M. J Chem Phys 2009;131(16):161107.
[60] vandenAkker CC, Engel MFM, Velikov KP, Bonn M, Koenderink GH. J Am
Chem Soc 2011;133(45):18030e3.
[61] Fu L, Ma G, Yan ECY. J Am Chem Soc 2010;132(15):5405e12.
[62] Fu L, Liu J, Yan EC. J Am Chem Soc 2011;133(21):8094e7.
[63] Xiao D, Fu L, Liu J, Batista VS, Yan EC. J Mol Biol 2011.
[64] Engel MF, Vandenakker CC, Schleeger M, Velikov KP, Koenderink GH,
Bonn M. J Am Chem Soc 2012;134(36):14781e8.
[65] Meng F, Abedini A, Plesner A, Verchere CB, Raleigh DP. Biochemistry
2010;49(37):8127e33.
[66] Diesner MO, Welle A, Kazanci M, Kaiser P, Spatz J, Koelsch P. Biointerphases
2011;6(4):171e9.
[67] Kurouski D, Deckert-Gaudig T, Deckert V, Lednev IK. J Am Chem Soc
2012;134(32):13323e9.
[68] Hamm P, Zanni MT. Concepts and methods of 2D infrared spectroscopy 2011.
[69] Cervetto V, Helbing J, Bredenbeck J, Hamm P. J Chem Phys 2004;121(12):
5935e42.
[70] Bredenbeck J, Helbing J, Nienhaus K, Nienhaus GU, Hamm P. Proc Natl Acad
Sci U S A 2007;104(36):14243e8.
[71] Strasfeld DB, Ling YL, Gupta R, Raleigh DP, Zanni MT. J Phys Chem B
2009;113(47):15679e91.
[72] Shim SH, Zanni MT. Phys Chem Chem Phys 2009;11(5):748e61.
[73] Wang L, Middleton CT, Singh S, Reddy AS, Woys AM, Strasfeld DB, et al. J Am
Chem Soc 2011;133(40):16062e71.
[74] Baiz CR, Peng CS, Reppert ME, Jones KC, Tokmakoff A. Analyst 2012;137(8):
1793e9.
[75] Zhuang W, Hayashi T, Mukamel S. Angew Chem Int Ed Engl 2009;48(21):
3750e81.
[76] Luca S, Yau WM, Leapman R, Tycko R. Biochemistry 2007;46(47):13505e22.
[77] Paravastu AK, Leapman RD, Yau WM, Tycko R. Proc Natl Acad Sci U S A
2008;105(47):18349e54.
[78] Shim SH, Gupta R, Ling YL, Strasfeld DB, Raleigh DP, Zanni MT. Proc Natl Acad
Sci U S A 2009;106(16):6614e9.
[79] Strasfeld DB, Ling YL, Shim SH, Zanni MT. J Am Chem Soc 2008;130(21):
6698e9.
[80] Ling YL, Strasfeld DB, Shim SH, Raleigh DP, Zanni MT. J Phys Chem B
2009;113(8):2498e505.
[81] Meng F, Abedini A, Plesner A, Middleton CT, Potter KJ, Zanni MT, et al. J Mol
Biol 2010;400(3):555e66.
[82] Cao P, Meng F, Abedini A, Raleigh DP. Biochemistry 2010;49(5):872e81.
[83] Knowles TP, Buehler MJ. Nat Nanotechnol 2011;6(8):469e79.
[84] KetenS, Buehler MJ. Comput Meth Appl Mech Eng 2008;197(41e42):3203e14.
[85] Jones OG, Mezzenga R. Soft Matter 2012;8(4):876e95.
[86] Xu Z, Paparcone R, Buehler MJ. Biophys J 2010;98(10):2053e62.
[87] Mucke N, Klenin K, Kirmse R, Bussiek M, Herrmann H, Hafner M, et al. PLoS
One 2009;4(11):e7756.
[88] Brangwynne CP, Koenderink GH, Barry E, Dogic Z, MacKintosh FC, Weitz DA.
Biophys J 2007;93(1):346e59.
[89] Relini A, Torrassa S, Ferrando R, Rolandi R, Campioni S, Chiti F, et al. Biophys J
2010;98(7):1277e84.
[90] Adamcik J, Jung JM, Flakowski J, De Los Rios P, Dietler G, Mezzenga R. Nat
Nanotechnol 2010;5(6):423e8.
[91] Gosal WS, Morten IJ, Hewitt EW, Smith DA, Thomson NH, Radford SE. J Mol
Biol 2005;351(4):850e64.
[92] Sachse C, Grigorieff N, Fandrich M. Angew Chem Int Ed 2010;49(7):1321e3.
[93] MudgalP, Daubert CR, Foegeding EA. Food Hydrocolloids 2009;23(7):1762e70.
[94] Aymard P, Nicolai T, Durand D, Clark A. Macromolecules 1999;32(8):2542e52.
[95] Veerman C, Ruis H, Sagis LM, van der Linden E. Biomacromolecules
2002;3(4):869e73.
[96] Jordens S, Adamcik J, Amar-Yuli I, Mezzenga R. Biomacromolecules
2011;12(1):187e93.
[97] Ban T, Hamada D, Hasegawa K, Naiki H, Goto Y. J Biol Chem 2003;278(19):
16462e5.
[98] Castro CE, Dong J, Boyce MC, Lindquist S, Lang MJ. Biophys J 2011;101(2):
439e48.
[99] Kellermayer MS, Grama L, Karsai A, Nagy A, Kahn A, Datki ZL, et al. J Biol
Chem 2005;280(9):8464e70.
[100] Karsai A, Martonfalvi Z, Nagy A, Grama L, Penke B, Kellermayer MS. J Struct
Biol 2006;155(2):316e26.
[101] Alsteens D, Ramsook CB, Lipke PN, Dufrene YF. ACS Nano 2012;6(9):7703e11.
[102] Ganchev DN, Cobb NJ, Surewicz K, Surewicz WK. Biophys J 2008;95(6):
2909e15.
[103] Tanaka M, Collins SR, Toyama BH, Weissman JS. Nature 2006;442(7102):
585e9.
[104] Kroes-Nijboer A, Venema P, Baptist H, van der Linden E. Langmuir
2010;26(16):13097e101.
[105] Govaerts C, Wille H, Prusiner SB, Cohen FE. Proc Natl Acad Sci U S A
2004;101(22):8342e7.
[106] Kishimoto A, Hasegawa K, Suzuki H, Taguchi H, Namba K, Yoshida M.
Biochem Biophys Res Commun 2004;315(3):739e45.
[107] Perutz MF, Finch JT, Berriman J, Lesk A. Proc Natl Acad Sci U S A 2002;99(8):
5591e5.
[108] van Mameren J, Vermeulen KC, Gittes F, Schmidt CF. J Phys Chem B
2009;113(12):3837e44.
[109] Collet JP, Shuman H, Ledger RE, Lee S, Weisel JW. Proc Natl Acad Sci U S A
2005;102(26):9133e7.
[110] Loveday SM, Rao MA, Creamer LK, Singh H. J Food Sci 2009;74(3):R47e55.
[111] Akkermans C, van der Goot AJ, Venema P, van der Linden E, Boom RM. Food
Hydrocolloids 2008;22(7):1315e25.
[112] Krysmann MJ, Castelletto V, Kelarakis A, Hamley IW, Hule RA, Pochan DJ.
Biochemistry 2008;47(16):4597e605.
[113] LovedaySM, Su JH, Rao MA, Anema SG, Singh H. Int Dairy J 2012;26(2):133e40.
[114] Veerman C, de Schiffart G, Sagis LM, van der Linden E. Int J Biol Macromol
2003;33(1e3):121e7.
[115] Bolisetty S, Harnau L, Jung JM, Mezzenga R. Biomacromolecules 2012;13(10):
3241e52.
[116] Corrigan AM, Donald AM. Langmuir 2009;25(15):8599e605.
[117] Valentine MT, Kaplan PD, Thota D, Crocker JC, Gisler T, Prud’homme RK, et al.
Phys Rev E Stat Nonlin Soft Matter Phys 2001;64(6 Pt 1):061506.
[118] GosalWS, Clark AH, Ross-Murphy SB. Biomacromolecules 2004;5(6):2408e19.
[119] Corrigan AM, Donald AM. Eur Phys J E Soft Matter 2009;28(4):457e62.
[120] Koenderink GH, Atakhorrami M, MacKintosh FC, Schmidt CF. Phys Rev Lett
2006;96(13):138307.
[121] Atakhorrami M, Mizuno D, Koenderink GH, Liverpool TB, MacKintosh FC,
Schmidt CF. Phys Rev E Stat Nonlin Soft Matter Phys 2008;77(6 Pt 1):061508.
[122] Boulet-Audet M, Vollrath F, Holland C. Phys Chem Chem Phys 2011;13(9):
3979e84.
M. Schleeger et al. / Polymer xxx (2013) 1e16 15
Please cite this article in press as: Schleeger M, et al., Amyloids: From molecular structure to mechanical properties, Polymer (2013), http://
dx.doi.org/10.1016/j.polymer.2013.02.029
Michael Schleeger received his diploma in chemistry from
the University of Bonn (2004). For his Ph.D. thesis, he
joined the group of Joachim Heberle at Bielefeld Univer-
sity, where he received his Ph.D. degree in 2009. After 2
years of postdoctoral research in the Department of
Physics at the FU Berlin, he joined the group of Prof.
Mischa Bonn at the Max-Planck Institute for Polymer
research in Mainz as a research fellow in 2012. His current
research focus is on the structure and interactions of am-
yloids based on vibrational spectroscopy, in particular
sum frequency generation spectroscopy.
Corianne van den Akker obtained her Master’s degree in
Biomedical Engineering from the University of Twente,
The Netherlands in 2010. She is currently studying for a
PhD with Prof. Gijsje Koenderink at FOM Institute AMOLF
in Amsterdam, with research orientated towards the me-
chanical properties and molecular conformation of amy-
loid fibrils.
Dr. Tanja Deckert-Gaudig works as a postdoc in the
research group of Prof. V. Deckert at the Institute of Pho-
tonic Technology in Jena. She obtained her Ph.D. in Organic
Chemistry in 1997 with Prof. S. Hünig at the University of
Würzburg. After a maternal leave she started working on
TERS with Prof. Deckert at the University of Dresden in
2002, followed by the Institute of Analytical Sciences in
Dortmund. Since 2009 she works in Jena, where her main
interest is the structural elucidation of biopolymers on the
nanoscale by TERS.
Prof. Volker Deckert holds a joint position at the Institute
of Physical Chemistry at the University of Jena, Germany
and the Institute of Photonic Technology, also in Jena. He
obtained his Diploma and Ph.D. from the University of
Würzburg, Germany, working on difference-Raman spec-
troscopy. As a postdoc at the University of Tokyo and the
Kanagawa Academy of Science in Kawasaki, he worked
on non-linear and time-resolved laser spectroscopy of
photo-induced isomerisation reactions . During his habili-
tation at the ETH Zurich he started working on the devel-
opment of high spatial resolution techniques for Raman
spectroscopy. This topic was the basis of his next positions
at the TU Dresden, ISAS Dortmund and now in Jena, where
he in particular explores the possibilities of the technology
to investigate structural changes of bio-related compounds
with nanometer resolution.
Dr. Krassimir Velikov is a Science/Team Leader in Unilever
R&D Vlaardingen. He received his PhD from the University
of Utrecht, The Netherlands. His main research interests
cover topics of soft-condensed ma tter, se lf-as sembly,
colloid and interface science of dispersions (e.g. suspen-
sions, foams, emulsions) and their uses to control product
functionality (e.g. stability, appearance, texture), physical-
chemistry of digestion, and formulation and delivery of
functional ingredients. He is an adjunct assistant professor
in the Soft Condensed Matter group at the Debye Institute
for NanoMaterials Science, Utrecht University and a special
adjunct associate professor in the Department of Chemical
and Biomolecular Engineering in the College of Engineer-
ing, North Carolina State University (USA). He is a Program
Director of Molecular Structure of Food program in
NanoNextNL.
Gijsje Koenderink received her Ph.D. from Utrecht Uni-
versity in 2003 in the area of physical and colloid chem-
istry. She conducted postdoctoral research at the VU
University (Amsterdam) and Harvard University (Cam-
bridge MA) on biophysical properties of cytoskeletal bio-
polymers. She joined the FOM Institute AMOLF in
Amsterdam as a group leader in 2006, where she started
the group “Biological Soft Matter”. Her research interests
focus on the structural and mechanical properties of bio-
polymers, the non-equilibrium properties of the cellular
cytoskeleton, and cellular mechanosensing.
Mischa Bonn (January 25, 1971, The Netherlands) received
his PhD in 1996 for work resulting from a collaboration
between the Technical University of Eindhoven and the
FOM Institute for Atomic and Molecular Physics, AMOLF.
After post-doctoral work at the Fritz-Haber Institute in
Berlin and Columbia University in New York, Mischa
worked at the chemistry department at Leiden University
(1999e2004), before moving to AMOLF (2004) as head of
the “Biosurface Spectroscopy”group. Mischa received
2009 the Gold Medal from the Royal Dutch Chemical Soci-
ety. In 2011 Mischa was appointed as one of the Directors
of the Max Planck Institute for Polymer Research in Mainz.
His research is centered around laser-based vibrational
spectroscopies, specifically surface and THz spectroscopies
and CARS microscopy.
M. Schleeger et al. / Polymer xxx (2013) 1e1616
Please cite this article in press as: Schleeger M, et al., Amyloids: From molecular structure to mechanical properties, Polymer (2013), http://
dx.doi.org/10.1016/j.polymer.2013.02.029
