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Large-scale polymorphism and auto-catalytic effect in insulin fibrillogenesis

Vito Foder
a,*ab Marco van de Weertb and Bente Vestergaarda

Published on 03 August 2010. Downloaded by New Copenhagen University on 10/06/2013 09:01:23.

Received 28th March 2010, Accepted 22nd June 2010

DOI: 10.1039/c0sm00169d
Under specific conditions the protein hormone insulin is prone to form amyloid fibrils and is widely
used as a model system to study fibril formation mechanisms. In this work we studied thermally
induced fibril formation of insulin in acidic solutions. We mainly focused on the effect of the initial
protein concentration on the final fibril morphology and the connection between morphology and
secondary nucleation mechanisms. Atomic force microscopy (AFM) analysis revealed a significant
fibril polymorphism on the macroscopic level dependent on protein concentration, ranging from thin
and elongated fibrils to highly complex superstructures. Moreover, amyloid superstructures formed at
high insulin concentration resulted in a lower propensity in being stained by Thioflavin T (ThT) and,
for these structures, a reduced catalytic effect in seeding experiments was also detected. These results
suggest a crucial involvement of the macroscopic properties of fibril surfaces in determining secondary
nucleation pathways in insulin fibrillation.

Introduction
Amyloid fibril formation is a focal area in current biomedical
research. Understanding the mechanisms of the conversion from
the native protein state to the amyloidal states represents
a fundamental step to improve the purification, storage and
delivery of protein-based drugs.1 Moreover, investigating the
origin of high impact neurodegenerative diseases like Alzheimers, Parkinsons and type-II diabetes can not be separated from
an accurate description of the inter- and intra-molecular interactions involved in the formation of such amyloid aggregates.2,3
In fact, amyloid fibrils are recognized to be connected with the
onset of such diseases, although their role is still a matter of
debate. Fibrils show common structural properties with a specific
arrangement of b-sheets commonly known as a cross b structure
revealing a well defined X-ray fibre diffraction pattern, as well as
a typical birefringence activity and/or fluorescence emission
when bound to specific dyes.4 Moreover, under suitable conditions formation of such assemblies may also be induced in vitro
for a large number of proteins.
As a consequence, the capability for the native state to convert
into non-native aggregates is considered a general pathway in the
free energy landscape of a protein.5,6
Formation of the mature amyloid fibrils is determined by
a complex and diversified interconnection of a high number of
interactions appearing with different time and length scales. It is
commonly accepted that a partial destabilization of the native
state is a conditio sine qua non for the actual growth of amyloid
a
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences,
University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen ,
Denmark
b
Department of Pharmaceutics and Analytical Chemistry, Faculty of
Pharmaceutical Sciences, University of Copenhagen, Universitetsparken
2, DK-2100 Copenhagen , Denmark
Present address: Sector of Biological and Soft Systems, Dept. of
Physics, Cavendish Laboratory, University of Cambridge, JJ Thomson
Avenue, Cambridge CB3 0HE, UK. E-mail: vf234@cam.ac.uk, Tel:
+44 (0) 1223 337290. Robinson College, Grange Road, Cambridge
CB3 9AN, UK.

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aggregates.6,710 After that, a two-step reaction known as the

nucleation-elongation process occurs, with formation of an
assumed high energy species (nucleus) and subsequent elongation
by addition of either monomeric or multimeric non-native
protein.11,12 The growth kinetics may involve simple addition of
non-native protein to the nucleus (primary nucleation), or highly
cooperative mechanisms involving auto-catalysis (secondary
nucleation).1214 In the presence of secondary nucleation (fragmentation, branching or nucleation on the fibril surface) the
growth rate of the amyloid aggregates depends on the amount of
fibrils already present in the sample, and an exponential increase
of the fibril mass as a function of the reaction time occurs.12 As
a consequence, interactions between existing fibrils, native and
non-native protein in solution may in principle lead to both
microscopic15 and macroscopic polymorphism in fibril structures.1619 Thus, fibrils may generally conserve their basic structural
arrangement of cross b-sheet, yet experience different packing into
three dimensional superstructures.1921 Such polymorphism is not
only reported for in vitro fibril formation and represents a poorly
understood aspect of in vivo fibril formation.2225 Structurally
different fibrils or ensembles of fibrils can have different biophysical effects, varying solution properties and different biological
activities. Therefore it is crucial to understand the molecular
mechanism behind this polymorphism.20
Insulin is a model protein commonly used in the study of fibril
formation.26,27 In solution, insulin exists as a mixture of different
self-association states, dependent on the physico-chemical
parameters of the solution. Under specific conditions, i.e. low pH
and high temperature, the protein rapidly converts into amyloid
fibrils through three observable stages, which are a lag phase, an
exponential fibril growth phase and a final plateau as revealed by
different experimental techniques.2628 Fibrillation pathways for
insulin have been extensively studied both with respect to suggested underlying mechanisms and, more recently, structural
studies of the occurring species in solution during the fibrillation
process,29,30 making insulin a suitable model to study particular
aspects of protein aggregation.31 Recently, several reported
results support the idea of the involvement of secondary
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nucleation pathways in insulin fibril formation.19,28,3234 A key

role seems to be played by the fibril surfaces, which catalyze the
overall aggregation reaction.32 Several different insulin fibrillar
morphologies have been reported in the past, ranging from
straight and elongated fibrils, through spherulites,35 branched
and twisted fibrils31 to superstructures of fibrils.16,18 The balance
between the above mentioned species is highly sensitive to pH,36
cosolvent addition37,38 and pressure.37 Moreover, in vitro insulin
fibril formation is affected by an intrinsic stochasticity of the
process32 and also by an extreme sensitivity to sample handling
(e.g., centrifugation, filtering, cuvette materials and surfacevolume ratio). As for the majority of the amyloidogenic proteins,
this sensitivity makes the values for nucleation time and the
consequent growth rates of aggregates not absolute and difficult
to compare between different and independent investigators.
With the aim of studying how different initial insulin
concentrations may affect the final fibril morphologies, we
present an experimental study on thermally induced fibril
formation in bovine insulin samples in acidic conditions. An
investigation by AFM was performed at low (1 mg/ml) and high
(10 mg/ml) protein concentrations. Because of its chemical and
optical stability under the chosen experimental conditions (high
temperature and low pH),39 in situ Thioflavin T staining was used
for detection of the fibrillation process. In particular,
a pronounced difference in the capability of differently packed
fibril structures to bind to Thioflavin T was observed. Sonication
of these fibrils, subsequent fluorescence analysis and seeding
experiments provide insights into the mode of ThT-fibril binding
and shed light on the relation between secondary nucleation
mechanisms and fibril morphologies in insulin fibrillation.

added to each well prior to incubating the plate and the emission
intensity at 480 nm was recorded upon excitation at 450 nm. The
emission signal was detected from the bottom of the plate every
400 s by an optical fibre system (bottom-bottom configuration).

Atomic force microscopy

AFM analysis was carried out in air using a Nanoscope 3
workstation (Veeco). The tapping mode tips were from Olympus
AC160TS (OTESPA). During scanning, the 160 mm-long cantilever, with a nominal spring constant of 42 N/m, oscillated at its
resonance frequency (300 kHz). Height and amplitude images
were collected by capturing 512  512 points in each scan and the
scan rate was maintained below 1 line per second. AFM
measurements were carried out on fibrils formed after incubation
at 45  C for 24 h of bovine insulin at 1 mg/ml and 10 mg/ml (20%
acetic acid, NaCl 0.5 M pH 1.8) in an eppendorf tube without
stirring. Aliquots were taken out after gently tipping the tube to
ensure a homogeneous sample. Samples of equal protein
concentration were obtained by diluting 50 times (1 mg/ml
sample) and 500 times (10 mg/ml sample) in 20% acetic acid. 8 ml
were positioned on a freshly cleaved mica surface (1cm  1cm)
and a nitrogen flux was used for 5 min to dry the specimen.
Images were acquired at room temperature from several sample
areas to ensure that heterogeneous sample deposition did not
influence the results. AFM images were also created from fibril
samples after sonication (see the following Section).

Sonication of fibrils and seeding experiments

Materials and methods

Sample preparation and Thioflavin T experiments
Bovine insulin was purchased from Sigma Aldrich (cat. No.
I8500) and used without any further purification. All the samples
used in this study were prepared by dissolving bovine insulin in
20% acetic acid, NaCl 0.5 M (pH 1.8). To avoid formation of salt
crystals, the solvent was freshly prepared before each experiment
and filtered through 0.22 mm filters (MS 16534, Sartorius), before
adding bovine insulin. All samples at different concentrations
were singly prepared immediately prior to each experiment.
Protein concentration was determined by UV absorbance at 276
nm using an absorbance value of 1.0 for 1.0 mg/ml.40 Thioflavin
T (ThT) was purchased as the chloride salt from SigmaAldrich
Chemie GmbH (Schnelldorf, Germany). The purchased ThT was
recrystallized three times in demineralised water and the dye
concentration in water was estimated by Vis absorbance using
a molar extinction coefficient of 36,000 M 1 cm 1 at 412 nm.41
For in situ ThT fluorescence, experiments were carried out using
a plate reader system (Fluostar, BMG Labtech) with 96-microwell polystyrene plates (Nalge Nunc) with 200 ml of solution per
well and four replicates per sample. The plates were covered with
non-sterile Polyolefin sealing tape (Nalge Nunc) to avoid evaporation of the sample and incubated at 45  C without mechanical
shaking. A stock solution of ThT in Milli-Q water (1 mM) was
prepared and stored a 4  C protected from light to avoid photobleaching. ThT at the desired concentration (20 mM) was
4414 | Soft Matter, 2010, 6, 44134419

Fibril formation was induced in an eppendorf tube on a 1 mlsample of bovine insulin (10 mg/ml) in 20% acetic acid, NaCl 0.5
M (pH 1.8) and incubated 24 h at 45  C. After this, samples were
equilibrated to room temperature. Fibril-containing aliquots
were then diluted (10% v/v) in five eppendorf tubes containing
20% acetic acid. Sonication was performed on four out of five
samples using a duty cycle of 50%, output control of 1 in a pulsed
regime. The tip was carefully cleaned by multiple flushing with
ethanol and Milli-Q water before each immersion in the samples.
The extent of fibril break-up was varied by using different
numbers of consecutive pulses (1, 4, 6 and 12 pulses) for different
solutions. Importantly, in this regime of soft sonication, no
temperature increase was detected in the samples. ThT fluorescence (ThT 20 mM) was measured for 24 h at 30  C on 200 mlaliquots of the sonicated and unsonicated samples using the
already mentioned procedure for fluorescence detection.
Aliquots of the same samples at different sonication treatments
were also used for seeding experiments by adding the fibrils to
a fresh solution of bovine insulin 1 mg/ml in insulin in 20% acetic
acid, NaCl 0.5 M (pH 1.8) to a final concentration of 1% wfibril/
wnative. Afterward, such seeded solutions were incubated at 45  C
with 20 mM ThT and the temporal evolution of the ThT fluorescence was detected as described before. Finally, AFM analysis
was also carried out on the aliquots of sonicated samples as
described in the previous section. The whole procedure was
repeated 3 times on independent samples to check the reproducibility of all of the results.
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Results and discussion

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Polymorphism as a function of protein concentration

An AFM analysis of fibril morphologies formed at high (10 mg/
ml) and low (1 mg/ml) insulin concentration is presented in Fig. 1
and Fig. 2, respectively. For the fibrils obtained at high insulin
concentration (Fig. 1), scanning of several areas of the whole mica
surface after fibril deposition evidences a strong spatial heterogeneity in the distribution of fibrils. Wide areas of the mica surface
do not show the presence of any protein assembly (not shown) and
fibrils only appear in some zone with a high local concentration
(Fig. 1a). The insulin fibrils resemble the well known straight
template of most amyloid fibrils (Fig. 1b) with segments up to 1
mm. Interestingly, together with these thin structures, large and
highly packed bundles are present (dotted white circles in Fig. 1a).
Zooming in on these structures (Fig. 1b and 1c) reveals assemblies
with a diameter of 700 nm apparently formed by a large number
of intertwined fibrils. Moreover, other highly dense areas present
an increased complexity of the fibrillar topology (Fig. 1d) with
thin and very long fibrils showing general tendencies to branch,
twist and bend over a mesoscopic scale (Fig. 1e). In contrast,
images of fibrils obtained from the sample at 1 mg/ml (Fig. 2) show
a substantial presence of elongated structures homogeneously
distributed along the whole mica surface (Fig. 2a and 2b) with
only a weak tendency of clustering (arrows in Fig. 2c) and
branching (arrows in Fig. 2d and 2e). Such filaments were
observed to vary in length ranging from few hundred nm (Fig. 2c)
to 3 mm (Fig. 2d and 2e). It is worth to note that AFM observations for the two different concentrations have been carried out on
the same total amount of protein and using exactly the same
procedure for the deposition of the protein on the mica surface
(see Materials and Methods). This operation guarantees that the
differences between images in Fig. 1 and 2 may only be ascribed to
the intrinsic morphologies of the fibrils and not to the experimental approach.

According to previous reports, different environmental factors

may affect the aggregation mechanisms as well as the spatial
arrangement of the fibrils, leading to different fibril morphologies.17,18,37,38 Further, secondary nucleation pathways have been
suggested to play a main role in the insulin fibrillation reaction4244
leading to an exponential temporal course for the fibril
growth.18,19,3133
Fig. 1 and Fig. 2 suggest the involvement of such pathways in
determining the macroscopic features of the final amyloidal
structures, depending on the initial protein concentration. In
fact, formation of early stable aggregates in a given region of the
sample can be considered intrinsically stochastic and spatially
localized in the samples28 and because of the secondary pathways, new fibrils are very prone to grow close to such early stable
aggregates.31,34 At 10 mg/ml, the local monomer concentration
around the first few single fibrils is very high. We suggest that
fibril surfaces may enhance and speed up the unfolding of
monomers, and that this effect is more pronounced at such high
concentrations. This in turn leads to a fast nucleation and growth
of new fibrils close to the already formed fibrils (i.e. secondary
nucleation).13,31 The described phenomenon readily propagates
throughout the sample hence determining the overall complex
topology observed (Fig. 1). On the other hand, at 1 mg/ml the
reduced local monomer concentration in proximity of the early
fibrils reduces the influence of fibril surfaces on the early
unfolding events and hence there is a lowered tendency of the
consequent fibril clustering. In the latter case, even though the
secondary pathways still cause an exponential temporal course,32
unfolding and nucleation in the bulk solution can be effective in
contributing to an overall more independent growth of fibrils
without pronounced clustering. As a consequence, the elongation
leading to the straight- and elongated-like fibrils with a reduced
clustering effect is more probable at lower concentration (Fig. 2).
It is also worth to note that the occurrence of a low number of
clusters (Fig. 2c and 2e) is in accordance with this suggestion,

Fig. 1 AFM amplitude scans after fibril formation from 10 mg/ml bovine insulin in 20% acetic acid, 0.5 NaCl, after 24 h of incubation at 45  C. (a)
10mm scan size and relative zooms at (b) 5 mm and (c) 1.6 mm. (d) 5 mm scan size of another area of the mica surface and (e) relative zoom at 1.25 mm.

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Fig. 2 AFM amplitude scans after fibril formation of 1 mg/ml bovine insulin in 20% acetic acid, 0.5 NaCl, after 24 h of incubation at 45  C. (a) 15mm
scan size and relative zooms at (b) 5 mm and (c)1.25 mm. (d) 3 mm scan size of another area of the mica surface and (e) relative zoom at 1.25 mm. White
arrows indicate clustering effect (Figure 2c) and branching (Figure 2e).

being the surface of single individual fibrils likely the same at

both high and low concentrations. Interestingly, Loksztejn and
Dzwolak recently used AFM to show a transition from single
dispersed fibrils to a massive fibril network when insulin solutions were agitated.33 In that study, agitation probably affects the
balance between primary and secondary nucleation. We suggest
that this is because shaking will cause a greater diffusion of early
stable insulin fibrils in the whole solution,28 hence a greater
probability that insulin molecules will be in close proximity of
fibril surfaces. In accordance with our theory, such mechanism
would lead exactly to the same effect as we described above for
high concentration samples, explaining the observed clustering
and laterally associated fibrils.33

previous Section, the difference in the fibril clustering at low (1

mg/ml) and high (10 mg/ml) insulin concentration could in
principle explain the saturation effect of the ThT fluorescence
shown in Fig. 3. In fact, this would be in accordance with existing
models of ThT-fibril binding,41,4547 claiming that a crucial role is
played by the accessible surface of the fibril providing binding
sites for the dye, i.e. that the ThT signal is mainly a measure of
the accessible fibril surfaces and not of the actual fibril mass.
Clustered fibrils will have a reduced accessible surface per unit
fibril mass as compared to the individual fibril structures mainly
detected at low concentration (Fig. 2). Hence, ThT staining
should be strongly affected by a more compact packing of fibrils
as the one observed in the current study (Fig. 1ce).

Morphology effect on ThT staining and autocatalysis

Using the same approach as previously described,32 in situ ThT
fluorescence as a function of incubation time was followed for
five insulin concentrations in the range 110 mg/ml at 45  C.
Confirming the previous results on human insulin,32 all the
kinetics obtained for bovine insulin present a characteristic
sigmoidal profile with a decreasing lag time at increasing protein
concentration (not shown). In Fig. 3 the ThT fluorescence final
value (FFV) at the end of these reactions is shown as a function
of the initial protein concentration. As also shown for human
insulin and with the same degree of reproducibility,32 the data
display a narrow range of linearity (<5mg/ml) and at higher
concentration the fluorescence signal does not noticeably
increase with further increases in protein concentration. In the
previous work, we suggested that the saturation effect shown in
Fig. 3 is likely to be a property of the fibrillation process itself,
and related to the different number of available ThT binding sites
at different protein concentrations. Based on the analysis of the
4416 | Soft Matter, 2010, 6, 44134419

Fig. 3 ThT fluorescence final value (FFV) as a function of bovine insulin

concentration (110 mg/ml) after incubation of the sample at 45  C in
20% acetic acid 0.5 M NaCl. Dashed line is a visual guide.

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If the above mentioned hypothesis is true, disrupting clusters

without dissolving individual fibrils should result in higher ThTfluorescence. Highly denaturant solvents such as guanidine-HCl
could in principle be used to obtain fibrils of smaller size.48
However, this procedure may result in a drastic change of the
chemical properties of amyloid fibril surface, possibly even
(partially) dissolving the amyloid structures. These changes in the
basic structure can strongly reduce the ThT staining.48 We
deliberately use sonication to produce monodispersed amyloid
fibrils of minimal and homogeneous size.49,50 This method
maintains the structural features of fibrils after fragmentation.50
Fibrils formed at 10 mg/ml of bovine insulin at 45  C have
been sonicated to different extent and in Fig. 4 the AFM images
for a sample after 6 pulses of sonication are shown as a representative example.The mica surface is homogeneously covered by
very short segments (Fig. 4a and 4b) and no significant differences are detected when comparing different areas of the sample
(data not shown). The observed segments are a few hundred nm
long (Fig. 4c) and no evidence of the complex superstructures as
the ones obtained for the unsonicated sample (Fig. 1) is present.
Images from other sonicated samples (1, 4 and 12 pulses) reveal
the same general features, i.e. disruption of big bundles (images
not shown). Fig. 5 shows the Thioflavin T fluorescence after
addition of the dye to the various sonicated samples. With
increasing number of sonication pulses, the fluorescence value
increases almost linearly reaching a constant value for sonication
pulses >4. Interestingly, the fluorescence value obtained after 12
pulses is 3 times higher than the value detected for an unsonicated sample. This difference is roughly of the same order as the
deviation from the linearity observed in Fig. 3 for the sample at
10 mg/ml. The experiments have been repeated for fibrils formed
at 1 mg/ml and 5 mg/ml. They reveal a lower increase in fluorescence value after 12 sonication pulses for 1 mg/ml (+25%) and

Fig. 4 AFM amplitude scans of fibril formed from 10mg/ml bovine

insulin in 20% acetic acid, 0.5 NaCl, after 24 h of incubation at 45  C
followed by a cycle of sonication (6 pulses). (a) 10mm scan size and
relative zooms at (b) 5 mm and (c) 1.6 mm. White arrows indicate short
fragments of fibrils obtained by sonication.

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Fig. 5 ThT fluorescence final value (FFV) for fibrils formed at 10 mg/ml
of bovine insulin in 20% acetic acid 0.5 M NaCl at 45  C, diluted 1 : 10 v/v
and treated with different sonication pulses. Error bars represent absolute deviations observed on four replicates. Dashed line is a visual guide.

a value 2.5 times higher for 5 mg/ml (data not shown). This
indirectly indicates that at low concentration fibril clustering and
the formation of complex fibril structures are greatly suppressed
compared to fibrils formed at high concentration. These findings
corroborate the AFM data (Fig. 2).
Our data (Fig. 4 and Fig. 5) suggest that sonication treatments
are able to disassemble the superstructures originally formed at
10 mg/ml, thereby making available a larger number of ThT
binding sites (Fig. 5), reaching a maximum number of available
sites for pulses >4. We conclude that fibril clustering, bundles
formation (dotted white circles in Fig. 1a) and the formation of
even larger superstructures are responsible for the saturation
effect observed in Fig. 3, in accordance with independent
reports.33
Finally, the catalytic effect on fibrillation by differently sonicated fibrils was studied. In Fig. 6a the fibrillation kinetics of
bovine insulin 1 mg/ml at 45  C with the seeds from differently
sonicated samples are shown and compared to the unseeded
kinetics. Clearly, the main effect of seeding a fresh solution is to
shorten the lag time51 only slightly varying the growth rate
(Fig. 6a). Interestingly, the seeding effect on fresh solutions is
dependent on the sonication treatment, i.e. on the amount of
available fibril surfaces. Fig. 6b shows the inverse of the time
necessary to reach the 50% of the kinetics (1/t50%) and this value
is highest when the seeds are significantly sonicated (>4, Fig. 6b).
The data in Fig. 6a and 6b suggest that when the fibril surfaces
are more accessible (as previously verified by the increase of the
ThT signal, Fig. 5) their catalytic effect is significantly enhanced.
Basing on the idea that ThT-fluorescence levels reflect the
available fibril surface area,41,4547 our results thus provide
evidence for a direct involvement of available fibril surface area
in secondary nucleation, i.e. heterogeneous nucleation.12
It is worth to note that the overall rate of fibril formation (1/
t50%) as a function of the insulin concentration shows a steep
linear increase up to 5mg/ml, but then remains relatively constant
at increasingly higher concentrations.32
The latter tendency has previously been ascribed to the nucleus
becoming stable in solution at concentration higher than
5mg/ml29,52 with an underlying assumption mainly based on
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accessible surface (Fig. 1), diminishing the efficiency of the on

going autocatalytic pathways (Fig. 6). The latter effect is probably dominant and abolishes the expected faster growth rate for
higher concentrations. In conclusion, specifically for insulin, we
believe that at high concentration the occurrence of fibril
morphologies with a pronounced clustering may explain the
weakening of concentration-dependence of the rate.

Conclusions

Fig. 6 (a) Kinetics of 1 mg/ml bovine insulin fibrillation in 20% acetic

acid, 0.5 M NaCl, 45  C. Samples were seeded with fibrils (1% wfibril/
wnative) from samples at 10 mg/ml and sonicated with different pulses
numbers. The ThT fluorescence is shown as a function of incubation time
after seeding with different stocks. No seeds (black circles), seeds not
sonicated (open circles), seeds sonicated with 1 pulse (black triangles),
seeds sonicated with 4 pulses (open triangles), seeds sonicated with 6
pulses (black square) and seeds sonicated with 12 pulses (open squares).
(b) Overall rate constant 1/t50% as a function of the sonication pulses as
calculated from the kinetics in Figure 6a. Statistical error for 1/t50% is
5% of the values.

a numerical simulation for a nucleated polymerization.52

However, there is a lack of experimental evidence for a concentration dependent accumulation of oligomers, and our data
provide an alternative interpretation for the typical appearance
of the plot of 1/t50% versus concentration. We suggest that, in the
range 05 mg/ml, i.e. under conditions where fibrils form with
larger accessible surface areas (Fig. 2), increasing the protein
concentration greatly stimulates the overall effect of the
secondary nucleation hence resulting in strong concentration
dependence for the rate. Above 5mg/ml, there are two separate
tendencies. Firstly, in the early stages of the process, higher
concentration would promote both a faster bulk nucleation and
an enhanced effect of secondary nucleation pathways.
Conversely, at the same time, the fibrils are formed with an
altered macroscopic morphology, i.e. clusters with a reduced
4418 | Soft Matter, 2010, 6, 44134419

We presented an experimental study of insulin fibril formation in

acetic acid solutions. Using AFM and Thioflavin T staining we
have revealed a pronounced macroscopic polymorphism for
amyloid fibrils dependent on initial protein concentration, with
fibril clustering and thin fibrils mainly occurring at high and low
concentration, respectively. Such difference in the morphology is
also related to a different propensity in creating a fluorescent
complex with ThT, with the clusters being less prone to be
stained proportional to the actual amount of fibril. The latter
effect can be reversed by disrupting the clusters via sonication.
We further showed by combined sonication and seeding experiments that the available fibril surface area is directly related to
the efficiency in catalyzing the aggregation reactions, which
strongly suggests that interaction between non-fibrillated protein
and the fibril surface itself probably promotes initial unfolding
and subsequent nucleation, i.e. heterogeneous nucleation.
In conclusion, our data clarify a technical aspect reported in
the recent literature18,32,33 about ThT efficiency in quantitative
staining of amyloidal species, indicating the packing of fibril as
a limiting factor for the emission activity. Moreover, most
importantly, the central conclusions presented in this work
represent a significant output relating basic molecular mechanisms to the fibril morphologies and the temporal features of
fibrillation. It is worth noting that recent studies reported
a potential link between fibril polymorphism and the consequent
pathological state.53 We speculate that the autocatalytic effect of
accessible fibril surface and a deep investigation of fibril surface
properties may offer an explanation for such occurring multiplicity of morphologies.

Acknowledgements
We wish to thank Sven Frokjaer, Minna Groenning and
Maurizio Leone for useful discussions and financial support.
Fabio Librizzi is acknowledged for seeding ideas behind this
work. We thank Tue Hassenkam and the Nano-Science Center,
University of Copenhagen for the availability of the atomic force
microscope. Vito Foder
a was partly supported by a Lifelong
Learning Program (LLP) - Erasmus Placement.

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