Báo cáo khoa học: Amyloid oligomers: spectroscopic characterization of amyloidogenic protein states docx

MINIREVIEW
Amyloid oligomers: spectroscopic characterization of
amyloidogenic protein states
Mikael Lindgren
1
and Per Hammarstro
¨
m
2
1 Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway
2 IFM-Department of Chemistry, Linko
¨
ping University, Linko
¨
ping, Sweden
Introduction
Amyloidosis manifests itself through the extracellular
deposition of insoluble protein fibrils, leading to tissue
damage and disease. The fibrils form when normally
soluble proteins and peptides misfold and self-associate
in an abnormal manner [1]. The mechanisms behind
the self-assembly of naturally occurring proteins into
Keywords
amyloid proteins; fluorescence
spectroscopy; oligomeric amyloid state;
prefibrillar intermediate state; real-time
detection
Correspondence
M. Lindgren, Department of Physics,
Norwegian University of Science and
Technology, Trondheim, Norway

Fax: +47 73597710
Tel: +47 73593414
E-mail:
(Received 4 September 2009, revised
14 December 2009, accepted 7 January
2010)
doi:10.1111/j.1742-4658.2010.07571.x
It is assumed that protein fibrils manifested in amyloidosis result from an
aggregation reaction involving small misfolded protein sequences being
in an ‘oligomeric’ or ‘prefibrillar’ state. This review covers recent optical
spectroscopic studies of amyloid protein misfolding, oligomerization and
amyloid fibril growth. Although amyloid fibrils have been studied using
established protein-characterization techniques throughout the years, their
oligomeric precursor states require sensitive detection in real-time. Here,
fluorescent staining is commonly performed using thioflavin T and other
small fluorescent molecules such as 4-(dicyanovinyl)- julolidine and
1-amino-8-naphtalene sulphonate that have high affinity to hydrophobic
patches. Thus, populated oligomeric intermediates and related ‘prefibrillar
structures’ have been reported for several human amyloidogenic systems,
including amyloid-beta protein, prion protein, transthyretin, a-synuclein,
apolipoprotein C-II and insulin. To obtain information on the progression
of the intermediate states, these were monitored in terms of fluorescence
parameters, such as anisotropy, and quantum efficiency changes upon
protein binding. Recently, new antibody stains have allowed precise moni-
toring of the oligomer size and distributions using multicolor labelling and
single molecule detection. Moreover, a pentameric thiophene derivative
(p-FTAA) was reported to indicate early precursors during A-beta
1-40
fibrillation, and was also demonstrated in real-time visualization of cerebral
protein aggregates in transgenic AD mouse models by multiphoton micros-

copy. Conclusively, molecular probes and optical spectroscopy are now
entering a phase enabling the in vivo interrogation of the role of oligomers
in amyloidosis. Such techniques used in parallel with in vitro experiments,
of increasing detail, will probably couple structure to pathogenesis in the
near future.
Abbreviations
AD, Alzheimer’s disease; ANS, 1-amino-8-naphthalene sulfonate; DCVJ, (4-(dicyanovinyl)-julolidine); LCO, oligomeric LCPs; LCP,
luminescent-conjugated polymers; p-FTAA, pentameric thiophene derivative; ThT, thioflavin T; TTR, transthyretin.
1380 FEBS Journal 277 (2010) 1380–1388 ª 2010 The Authors Journal compilation ª 2010 FEBS
amyloid deposits remain a mystery, even though it is
well known that the final fibrillar structures have a
number of structural properties in common, such as
the pronounced b-sheet secondary structure and
unbranched fibril morphology. Amyloids are associ-
ated with serious diseases, including systemic amyloi-
dosis, Alzheimer’s disease (AD), maturity-onset
diabetes and the prion-related transmissible spongi-
form encephalopathies [2,3]. Moreover, the common
fibrillar structures also have the ability to bind small
molecules such as the widely used amyloid stains thio-
flavin T (ThT) [4] and Congo red [5], with concomitant
alterations of their optical properties in terms of, for
example, fluorescence quantum efficiency and their
influence of polarized light rendering their appearance
birefringent. It is usually anticipated that a multi-order
reaction, such as the formation of amyloid fibrils,
involves thousands of monomeric protein molecules
and that it proceeds through the formation of interme-
diate smaller structures, at least transiently. There is
compelling evidence that the principal component

common to many of the related diseases, in addition
to the insoluble fibrillar deposits, are in the form of
populated prefibrillar small soluble aggregates [6],
these days referred to as ‘oligomers’ or ‘protofibrils’.
The properties and structures of such oligomeric aggre-
gates are of immense interest to understand amyloid
disease and amyloid formation. Furthermore, studies
in cell culture have revealed oligomers to show supe-
rior toxicity in relation to mature amyloid fibrils. For
example, oligomers of amyloid-b are known to impair
synaptic plasticity and to be toxic both in vivo and
in vitro [7,8]. Similar recent results on transthyretin
(TTR) [9] and insulin [10], have corroborated this to
be a common property of amyloidogenic oligomers, at
least in cell culture. Studies of the above-mentioned
proteins and of other proteins linked to human amy-
loidoses, including lysozyme [11] and prion protein
[12], have shown that oligomers transiently populate
during fibrillation and, under the correct circum-
stances, can be exclusively populated without further
conversion into amyloid fibrils. Data from several
groups have demonstrated that the conformational
conversion from a native protein towards a fibrillar
state is a phenomenon common to many proteins and
peptides, even for proteins of various organisms that
are not involved in amyloid disease states, for example
the N-terminal domain of HypF from Escherichia coli
[13] and human tissue factor [14]. Findings such as
these have led to the hypothesis that amyloid fibril for-
mation and amyloidogenic oligomerization is a generic

property of the polypeptide backbone [15] and that
this is not limited to specific sequences. Hence, it is
perplexing why only certain proteins cause amyloid
diseases. Nevertheless, despite the sequence indepen-
dence of misfolding and misassembly in vitro it is evi-
dent that sequence does play a major role in dictating
amyloid formation, from the perspective both of pro-
tein stability [16] and of sequence specificity for model-
ling aggregation propensity [17]. In conclusion, both
sequence-dependent and sequence-independent (i.e.
conformation-dependent) mechanisms appear to control
the assembly and toxicity of amyloid oligomers.
To clarify the mechanism of fibril formation and the
relation of the amyloidogenesis processes to numerous
amyloid diseases are challenging tasks. Many studies
have addressed these aspects over the past two decades
and they have become a major research field. Early
studies utilizing turbidity and sedimentation combined
with ThT binding provided data on the appearance of
high-molecular-weight aggregates [18] or on the disap-
pearance of soluble low-molecular-weight peptides [19].
Using combinations of size-exclusion chromatography,
electron microscopy and quasielastic light-scattering
spectroscopy, it was possible to distinguish intermedi-
ate structures in terms of dimers and protofibrils of
typical sizes of a few nm in diameter up to 100–
200 nm, respectively [20]. Real-time monitoring of
fibril growth is essential, but is also very technically
demanding. This article reviews a selection of spectro-
scopic techniques, predominantly fluorescence tech-

niques, recently used and developed to follow protein
misfolding, oligomerization and amyloid fibril growth
in real time. We cannot, for the sake of space, cover
this entire field, so will merely provide an overview of
the exciting progress made.
Structural methods for misfolded
proteins and amyloids
The desire to elucidate the structure and properties of
proteins has a long history, and techniques for pro-
tein-structure characterization cover a very wide range
of techniques. As proteins in native and other thermo-
dynamically stable states can be formed in relatively
large quantities, and even in crystalline form, NMR
and X-ray diffraction are traditionally used to provide
detailed atomic resolution 3D information of their
individual molecular conformations. However, the
studies of protein aggregates and amyloids require the
development of other techniques because the final
products, as well as intermediate structures, will occur
in small quantities and with structural diversity of
both conformation and size, and these systems are also
present at conditions far from equilibrium, making
it difficult to produce reproducible and accurate
M. Lindgren and P. Hammarstro
¨
m Spectroscopy of amyloid oligomers
FEBS Journal 277 (2010) 1380–1388 ª 2010 The Authors Journal compilation ª 2010 FEBS 1381
assessments of structure. The situation is, in some
respects, similar to the situation of studying protein
folding and dynamic protein–protein interactions, such

as in the case of chaperone action. Restricting us to
amyoidic structures, techniques such as NMR [21],
EPR [22], electron microscopy and atomic force
microscopy [23] and X-ray diffraction [24], have been
used with some success, as have optical techniques
such as CD and light scattering, as mentioned in the
Introduction. FTIR spectroscopy revealed that certain
vibration-frequency bands can be used to follow the
aggregation and to explore environmental properties,
such as pH, solvent and temperature effects on the
structure [25]. Bovine insulin was recently found to
form different aggregated structures controlled by
reducing agents; investigations with FTIR showed that
one type of filament, consisting of antiparallel beta-
sheets, was found to be nontoxic in cell cultures,
whereas parallel b-sheet-structured fibrils were found
to be toxic with a remarkably lower ThT fluorescence
for the filaments [10]. In contrast to methods for struc-
tural investigations of mature fibrillar structures, the
best-suited biophysical technique for obtaining high-
resolution structural information on defined oligomeric
species populated during fibrillation is small-angle
X-ray scattering. This was recently demonstrated for
insulin by Vestergaard et al. [26], where a conforma-
tionally defined oligomer was shown to be a building
block for mature fibrils. However, there are some prac-
tical constraints for small-angle X-ray scattering,
namely (a) exclusive equipment is necessary, (b) it
requires a high concentration of approximately 0.1 mm
of protein, (c) it requires a population of a few species

(preferably one to three) at the same time, (d) the sam-
ple must be stable for typically several minutes for
recording the necessary amount of raw data and (e) it
involves a tedious data-analysis process.
Fluorescence methods for capturing
the intermediate oligomeric state
To obtain information more rapidly, and to be able to
use low protein concentrations, fluorescence tech-
niques, in combination with labeling, offer high sensi-
tivity and measurement of a diversity of structural
aspects, which are dependent on the fluorescent probe
used. In Fig. 1 the most common techniques are
depicted graphically in a cartoon, which are discussed
in more detail below. ThT or thioflavin S, and Congo
red, are commonly used to detect amyloid deposits in
biopsies or in ex vivo postmortem samples [27], as
depicted in Fig. 1 (top row). Further modified versions
of these dyes have also been used as in vivo imaging
agents of amyloid deposits [28]. Derivatives of thioflav-
ins [29], Congo red derivatives [30] and oxazine-deriva-
tives [28] typically bind amyloid fibrils in the
nanomolar to micromolar range with multiple binding
sites. For more details and extensive references on
molecular ligands towards amyloid fibrils the reader is
referred to recent reviews on the topic [31,32]. In this
article the topic will be restricted to recent work using
advanced fluorescence techniques and probes that have
been used to detect oligomeric amyloid protein states.
Small molecular dyes, such as [4-(dicyanovinyl)-
julolidine] (DCVJ), as well as derivatives of 1-amino-

8-naphthalene sulfonate (ANS, Bis-ANS), have been
used for amyloid fibril detection, and are known to
bind to the fibrillar or prefibrillar states with dissocia-
tion constants typically in the micromolar range. We
have successfully used these molecules for recording
the kinetics of oligomerization of A-state (molten glob-
ule type) TTR, under low-pH conditions [33]. DCVJ
and ANS were compared with ThT as probes for the
oligomerization of TTR. DCVJ and ANS showed
good binding and fluorescence at acidic pH, whereas
ThT did not bind oligomers at this pH, requiring a pH
shift in the assay buffer. DCVJ proved efficient in the
kinetic assay and showed a reasonable binding affinity
to preformed early state oligomers of TTR (Fig. 2).
DCVJ did not bind to native tetrameric TTR, render-
ing this an interesting molecule for following the path-
way of tetramer dissociation, which was not performed
in the cited work. ANS, by contrast, was also found to
be a good probe for the kinetic assay and also had a
reasonable fluorescence lifetime (16 ns), but ANS binds
to the thyroxine-binding pocket of native TTR, which
would make measurements of dissociation kinetics
impractical. That both DCVJ and ANS responded
more efficiently than ThT in both the kinetic and to
the isolated TTR oligomers indicates that ThT is more
selective towards fibrils than towards oligomers.
Nevertheless, ThT can respond also to oligomers, and
ThT was recently used to follow the in vitro formation
of highly toxic soluble amyloid-b oligomers, which was
assisted by the chaperon prefoldin [34].

Fluorescence anisotropy is a well-known technique
used to obtain information of molecular and protein
sizes from the apparent rotation diffusion of fluores-
cent probes, usually attached to mutated proteins
(Fig. 1, second row). The technique has been widely
used in studies analysing the changes from unfolded to
folded protein structures of sizes typically up to 30–
50 kD. It was adopted for use in studies of the aggre-
gation of amyloid-b using a fluorescein marker
attached to a cysteine introduced at position 7 [35].
Typical time-resolved anisotropy decays, assayed over
Spectroscopy of amyloid oligomers M. Lindgren and P. Hammarstro
¨
m
1382 FEBS Journal 277 (2010) 1380–1388 ª 2010 The Authors Journal compilation ª 2010 FEBS
time, displayed a trace with fast initial and slow ‘floor’
raising as time progressed during incubation after the
initiation of oligomerization (i.e. the contribution of
the ‘residual anisotropy’ to the overall anisotropy
increased). Similarly, small fluorescent probes, such as
DCVJ, ANS and bis-ANS, were tested and compared
with ThT for studies of TTR aggregation [33], reveal-
ing a clear correlation between the progress of fibril
formation and an increase of the slow anisotropic
component as a result of the probe binding to increas-
ingly larger fibrillar structures. Recently, it was also
demonstrated that from the steady-state polarization
the oligomerization process of a-synuclein could be
followed, which preceded ThT fluorescence kinetics of
the same process [36]. The time-resolved anisotropy

studies also demonstrated [33] the shortcoming of the
anisotropy technique: it is physically impossible to
obtain information of rotational correlation times
somewhat longer than (typically three to five times)
the decay time of the fluorescent probes used. As fluo-
rescein and ANS have decay times that are shorter
than 5 and 20 ns, respectively, only the latter could
actually give an accurate determination of the size of
the native protein complex if it was larger than
approximately 50 kDa, but other information from the
changes of the anisotropy can be used quantitatively
to follow the initial process. In order to improve the
accuracy for larger oligomeric complexes, a single cys-
teine mutant version of TTR (C10A ⁄ A37C) was modi-
fied with the long-lived pyrene-methyl iodo-acetamide
fluorophore. The decay time of the pyrene-methyl
iodo-acetamide fluorophore is up to 150–200 ns,
depending on the solvent, and thus permits the deter-
mination of rotational correlation times well above
600 ns. This allowed us to determine the kinetics of
oligomerization of A-state TTR and was used to deter-
mine the size of a toxic oligomer determined to be
composed of 20–30 monomer units [9]. Pyrene label-
ling has also been used for structural packing studies
during protein aggregation and amyloid fibrillation.
The unusually long lifetime of pyrene can render the
formation of an excited state dimer (excimer), given
that two pyrene moieties are in proximity. For protein-
aggregation studies this approach was first used to
FibrillationFibrillation

Fig. 1. Cartoon giving an overview of the
fluorescence techniques used to follow and
quantify the amyloid fibrillation process.
M. Lindgren and P. Hammarstro
¨
m Spectroscopy of amyloid oligomers
FEBS Journal 277 (2010) 1380–1388 ª 2010 The Authors Journal compilation ª 2010 FEBS 1383
map an aggregation interface in the formation of solu-
ble oligomers of carbonic anhydrase [37] and was sub-
sequently used for Sup35 prions [38], glucagon [39]
and a-synuclein fibrillation [40]. The major drawback
of pyrene attachment has been regarded to be twofold:
the necessity of a cysteine-scanning approach; and the
intrinsic bulkiness and hydrophobicity of the pyrene
label.
Modern fluorescent techniques and
novel spectroscopic stains
By using two different labels simultaneously, a multi-
tude of possibilities are available to gain more infor-
mation on the aggregation processes (Fig. 1, third
row). Ryan et al. [41] studied the effect of short-chain
phospholipids (DHPC and DHPS) on amyloid fibrilla-
tion of human apolipoprotein C-II. Differences in
morphology for different fibrillation conditions were
found using Alexa488 C5 maleimide and Alexa594 C5
maleimide in conjunction. Fluorescence resonance
energy transfer thus gave rise to quenching of the
donor probe (Alexa488) during fibril formation. More-
over, the fluorescence anisotropy was found to increase
as the fluorescence yield decreased during fibril forma-

tion. Previously it had been reported that fibrillation is
stimulated by the presence of negatively charged lipid
surfaces. Here, it was demonstrated that a similar
effect was achieved by interaction with the hydropho-
bic fatty acyl chain. By using two excitation sources it
is possible to detect single and multiple fluorescence
events independently, thus discriminating single mole-
cules from small aggregates that are more likely to
contain two different fluorophores. Orte et al. studied
this phenomenon by using site-specific labelling of two
different Alexa dyes (488 and 647) introduced in an
experimental amyloidogenic protein composed of the
SH3 domain of PI3 kinase [42,43]. By mixing these
labelled proteins in a 50 : 50 ratio and following the
aggregation process, their data revealed that the for-
mation of oligomeric species proceeded quickly, and
then more slowly, and that the oligomers were con-
sumed as longer fibril structures evolved. From the
magnitude of the fluorescent signal it was also possible
to estimate the size distribution (by comparison with
the single molecule cases). The oligomeric aggregate of
the SH3-domain was composed of 38 ± 10 monomers.
Also, recent work using antibody labels allowed the
sensitive detection of amyloid-b oligomers in human
cerebrospinal fluid by combining flow cytometry and
fluorescence resonance energy transfer [44].
Luminescent-conjugated polymers (LCPs) have been
developed over the last few years for use in studies of
protein conformations. In contrast to sterically more
restricted amyloidotropic dyes, such as ANS, DCVJ,

thioflavins and Congo red, LCPs contain a twistable
conjugated polymeric backbone whose geometry alters
their spectroscopic properties [45–48]. The most widely
used LCP in amyloid research has been polythiophenes.
Noncovalent binding to proteins, including amyloid,
constrains the rotational freedom of LCPs, altering
their spectral properties in a conformation-dependent
AB
C
Fig. 2. (A) The chemical scheme for TTR
amyloid fibrillation from the unfolded mono-
meric state in low salt and acidic pH. (B)
Kinetics, as detected by normalized fluores-
cence, comparing the fluorescent probes
DCVJ, ANS and ThT for detection of early
oligomeric species during the fibril forma-
tion. From the amplitudes it is evident that
ThT also showed a very slow additional
phase that is not fully included in the time-
scale depicted above. (C) The apparent dis-
sociation constants differ equally to their
response time towards early oligomers. The
figure panels are redrawn from a previous
publication [33].
Spectroscopy of amyloid oligomers M. Lindgren and P. Hammarstro
¨
m
1384 FEBS Journal 277 (2010) 1380–1388 ª 2010 The Authors Journal compilation ª 2010 FEBS
manner. The optical fingerprint obtained from the
bound LCP reflects the conformational differences in

distinct protein aggregates, as depicted in Fig. 1, lower
row. This property has been used to discriminate prion
aggregates associated with different prion strains
[49,50], conformational heterogeneities in A-beta amy-
loid plaques in Alzheimer disease mouse models [51] and
morphologically different amyloid deposits in systemic
AL amyloidosis [52]. LCP luminescent staining technol-
ogy constitutes an important new tool for the analysis of
amyloid and prion complexes when analyzed using mod-
ern high-resolution spectral imaging techniques. Impor-
tantly, the multiphoton excitation capabilities of a few
LCPs provided excellent performance when compared
with imaging using conventional ‘single photon’ excita-
tion [53]. The fluorescence quantum yield of LCPs, in
the order of 5 – 10%, is lower than that for conventional
molecular high-efficiency fluorescence systems based
on, for example, fluorescein. However, the nonlinear
properties, in terms of two-photon excitation fluores-
cence efficiency, are comparable owing to a high
two-photon absorption cross-section in the range of
(4–10) · 10
)48
cm
4
Æs per photon [53]. This is approxi-
mately two orders of magnitude better than fluorescein
based on two-photon absorption per molecule. Never-
theless, compensating for the molecular size and nor-
malizing to molecular weight, the number is still larger
than or comparable to that of the fluorescein analogs

and compensates for the less-efficient quantum effi-
ciency. Interestingly, it was also found that multiphoton
excitation schemes in some instances gave complemen-
tary spectral signatures in terms of polarization and
spectral shifts, in addition to the results obtained using
single photon excitation of the very same LCP-stained
samples. Multiphoton excitation opens up new possibili-
ties, as excitation in the near infrared wavelength region
allows deeper penetration into tissue, and thus better
in vivo imaging [54,55]. It has still not been reported that
A
B
C
Fig. 3. (A) The chemical structure of the
LCO p-FTAA. (B) Kinetic traces of A-beta
1-40
and A-beta
1-42
fibril formation, comparing
p-FTAA and ThT fluorescence. p-FTAA
responds to early prefibrillar aggregates
before ThT. The conversion of A-beta
1-42
is
faster than for A-beta
1-40
and hence ThT and
p-FTAA show overlapping kinetic traces
(right traces). The asterisks indicate time
points for samples collected for transmis-

sion electron microscopy, with the
corresponding micrographs above the
kinetic traces. (C) Fluorescence micrographs
of cerebral amyloid plaques in a transgenic
mouse model of AD stained with p-FTAA
and conventional antibodies (6E10 anti-A-beta
and A11 anti-oligomer) against amyloid
components. In the top micrographs,
p-FTAA and 6E10 show complete colocaliza-
tion, and in the bottom micrographs there is
partial overlap, indicating that oligomer
distribution is localized to certain foci within
the plaque. Figure panels are redrawn from
a previous publication [56].
M. Lindgren and P. Hammarstro
¨
m Spectroscopy of amyloid oligomers
FEBS Journal 277 (2010) 1380–1388 ª 2010 The Authors Journal compilation ª 2010 FEBS 1385
any synthetic molecule can be used to image oligomeric
states formed in vivo; however, very recently, oligomeric
LCPs (LCO) were reported that can pass through the
blood–brain barrier and be used for in vivo studies of
plaque distribution in AD mouse models [56]. In vitro
studies, which were presented in the same paper using
the LCO pentameric thiophene derivative (p-FTAA)
(Fig. 3A), showed interesting results with regard to the
detection of early prefibrillar oligomeric states that were
nonthioflavinophilic (Fig. 3B). It was demonstrated that
p-FTAA detects both prefibrillar states as well as
mature amyloid fibrils. Further important evidence was

the identification of foci within amyloid-b plaques that
stained with an anti-oligomer Ig and which were
co-stained with p-FTAA, suggesting an irregular distri-
bution of different conformational states (oligomers and
fibrils) within the same senile plaque (Fig. 3C) [56].
There are still several aspects of LCP-like molecules
that are well worth exploring. Can the spectroscopic
properties of an LCP bound to a prefibrillar oligomer
be used as a marker for an on- or off-pathway species?
If the spectroscopic property (i.e. conformation) of the
bound LCP mimics that of the mature fibril, is it likely
that the building block is conformationally related (i.e.
a building block for the mature fibril). If, however, the
amyloid oligomer requires a substantial conforma-
tional change before the formation of an amyloid
fibril, the spectral property will be different. Markers
of this type will certainly be imperative for the basic
understanding of pathways (intermediates) of amyloid
fibril formation. These are also crucial for imaging
purposes, to irrefutably ascribe endogenous oligomeric
states a pathological role also in vivo.
Acknowledgements
The work was supported by the Swedish Foundation
for Strategic Research (to PH), the Swedish research
council (to PH) and the Knut and Alice Wallenberg
foundation (to PH). P.H. is a Swedish Royal Academy
of Science Research Fellow sponsored by a grant from
the Knut and Alice Wallenberg Foundation. Part of
this work was supported by the European Union FP7
HEALTH (Project LUPAS). We thank Peter Nilsson

for valuable comments on the manuscript.
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