Structure and assembly–disassembly properties of wild‐type

Special Issue Article
Received: 9 June 2010,
Revised: 15 September 2010,
Accepted: 15 October 2010,
Published online in Wiley Online Library: 2011
(wileyonlinelibrary.com) DOI:10.1002/jmr.1112
Structure and assembly–disassembly
properties of wild-type transthyretin amyloid
protofibrils observed with atomic force
microscopyy
Ricardo H. Pires a,b *, Maria J. Saraiva b,c, Ana M. Damas b,c **
and Miklós S. Z. Kellermayera ***
Transthyretin (TTR) is an important human transport protein present in the serum and the cerebrospinal fluid.
Aggregation of TTR in the form of amyloid fibrils is associated with neurodegeneration, but the mechanisms of
cytotoxicity are likely to stem from the presence of intermediate assembly states. Characterization of these
intermediate species is therefore essential to understand the etiology and pathogenesis of TTR-related amyloidoses.
In the present work we used atomic force microscopy to investigate the morphological features of wild-type (WT) TTR
amyloid protofibrils that appear in the early stages of aggregation. TTR protofibrils obtained by mild acidification
appeared as flexible filaments with variable length and were able to bind amyloid markers (thioflavin T and Congo
red). Surface topology and contour-length distribution displayed a periodic pattern of 15 nm, suggesting that the
protofibrils assemble via an end-binding oligomer fusion mechanism. The average height and periodic substructure
found in protofibrils is compatible with the double-helical model of the TTR amyloid protofilament. Over time
protofibrils aggregated into bundles and did not form mature amyloid-like fibrils. Unlike amyloid fibrils that are
typically stable under physiological conditions, the bundles dissociated into component protofibrils with axially
compacted and radially dilated structure when exposed to phosphate-buffered saline solution. Thus, WT TTR can form
metastable filamentous aggregates that may represent an important transient state along the pathway towards the
formation of cytotoxic TTR species. Copyright ß 2011 John Wiley & Sons, Ltd.
Keywords: transthyretin; atomic force microscopy; protofibril; periodicity; amyloid; fibrillogenesis; cytotoxic oligomers
* Correspondence to: R. H. Pires, Department of Biophysics and Radiation
Biology, Faculty of Medicine, Semmelweis University, Tűzoltó u. 37-47, Budapest IX, H1094, Hungary.
E-mail: [email protected]
INTRODUCTION
Amyloidoses encompass a wide spectrum of highly debilitating
disorders involving systemic pathological lesions, neurodegeneration and several other tissue-specific dysfunctions (Chiti and
Dobson, 2006). In these disorders, perturbation of the protein’s
native fold results in aggregation, which ultimately leads to
the formation of large deposits constituted mostly of amyloid
fibrils. A related misfolding process is thought to result in the
amyloidogenic conversion of transthyretin (TTR). TTR is a
homotetrameric protein synthesized in the liver and the choroid
plexus of the brain which is then exported to the blood serum
and the cerebrospinal fluid, respectively (Fleming et al., 2009).
Aggregation of TTR in the form of amyloid fibrils that deposit in
extracellular space leads to a condition known as senile systemic
amyloidosis in the case of wild type (WT) TTR (Westermark et al.,
1990), while mutations in the TTR sequence are often associated
with familial amyloidotic polyneuropathy (FAP) (Sousa and
Saraiva, 2003).
In the case of WT TTR and, similarly to other proteins, the
in vitro amyloid fibrillogenesis is often initiated by acidifying the
medium so as to mimic the lysosomal milieu (Colon and Kelly,
1992). The lysosome is a mildly acidic (pH 5) cytoplasmic vesicle
whose housekeeping activity of degrading subcellular structures
has been associated with amyloidogenic disorders (Nixon, 2007).
** Correspondence to: A. M. Damas, IBMC-Institute for Molecular and Cell
Biology; Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.
E-mail: [email protected]
*** Correspondence to: M. S. Z. Kellermayer, Department of Biophysics and
Radiation Biology, Faculty of Medicine, Semmelweis University, Tűzoltó u.
37-47. Budapest IX, H1094, Hungary.
E-mail: [email protected]
a R. H. Pires, M. S. Z. Kellermayer
Department of Biophysics and Radiation Biology, Faculty of Medicine,
Semmelweis University, Tűzoltó u. 37-47, Budapest IX, H1094 Hungary
b A. M. Damas, R. H. Pires, M. J. Saraiva
IBMC-Institute for Molecular and Cell Biology; Rua do Campo Alegre, 823,
4150-180 Porto, Portugal
c A. M. Damas, M. J. Saraiva
ICBAS-Instituto de Ciências Biomédicas de Abel Salazar, Universidade do
Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal
y
This article is published in Journal of Molecular Recognition as a focus on AFM
on Life Sciences and Medicine, edited by Jean-Luc Pellequer and Pierre Parot
(CEA Marcoule, Life Science Division, Bagnols sur Cèze, France).
Abbreviations: AFM, atomic force microscopy; CAC, circular autocorrelation; DFT,
discrete Fourier transform; FAP, familial amyloidotic polyneuropathy; FFT, fast Fourier
transform; nCAC, normalized circular autocorrelation; PBS, phosphate buffered
saline; pI, isoelectric point; ThT, thioflavin T; TTR, transthyretin; WT, wild type.
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R. H. PIRES ET AL.
The acidic environment leads to the disassembly of TTR’s
quaternary structure followed by partial unfolding of the
monomer that becomes prone to aggregation (Lai et al., 1996;
Foss et al., 2005). The acidification process has been proposed to
be sufficient for initiating the assembly of TTR amyloid fibrils
(Colon and Kelly, 1992). However, the process of fibrillogenesis
depends on various additional conditions as well, such as
temperature, protein concentration, ionic strength, agitation and
pH. Manipulation of some of these parameters has been shown to
lead to the appearance of various intermediate aggregation
states prior to the appearance of mature amyloid fibrils (Kodali
and Wetzel, 2007). These intermediate states have been receiving
increased attention because they possess much greater cytotoxic
potential than the fibrils (Caughey and Lansbury, 2003).
One of the amyloid fibrillogenic intermediate states is
the protofibril, which can be frequently found in diverse
amyloidogenic protein preparations and displays high cytotoxic
activity (Caughey and Lansbury, 2003). The term ‘protofibril’ is
used in the literature rather loosely, often referring to a wide
range of intermediates sometimes irrespective of their morphology. Here we adopt the terms ‘oligomer’, ‘protofibril’,
‘protofilament’, and ‘fibril’ as defined in a recent review (Kodali
and Wetzel, 2007). Accordingly, protofibrils are flexible linear
aggregates approximately 500 nm in length, and may (but usually
do not) contain a periodic substructure. Protofibrils may be the
direct precursors of protofilaments which, via a hierarchical
assembly process, give rise to amyloid fibril structure (Khurana
et al., 2003). In fact, in the case of the Aß1–40 peptide, the
presence of protofibril-specific antibodies in the aggregation
reaction effectively precludes the formation of mature amyloid
fibrils, leading to the accumulation of protofibrils (Habicht et al.,
2007). In contrast, in the case of ß2-microglobulin (ß2m), under
certain conditions the in vitro fibrillation reaction leads to the
accumulation of protofibrils without the formation of mature
amyloid fibrils (Kad et al., 2001; Gosal et al., 2005). Furthermore,
these protofibrils are unable to accelerate the assembly of
amyloid fibrils under mature fibril-forming conditions, suggesting
that they might be ‘off-pathway’ kinetic traps (Kad et al., 2001).
In the case of TTR the structural features of intermediate states
is relatively unexplored despite their high cytotoxic activity
(Sousa et al., 2001; Sousa et al., 2002). Detailed structural studies
have been carried out only down to the level of the TTR amyloid
protofilament for which two main models have been proposed.
One of them suggests that the protofilament is composed by a
single array of monomers (Inouye et al., 1998), that laterally
associate via a hierarchical assembly mechanism to form amyloid
fibrils (Cardoso et al., 2002). Another model proposes a double
helical structure containing an 11.55 nm repeat (Blake and
Serpell, 1996; Blake et al., 1996).
Since protofibrils have been proposed to direct precursors of
amyloid fibrils displaying cytotoxic activity, studies addressing
their structure and assembly dynamics are relevant in understanding their cytotoxic behavior as well as their role within
the amyloid aggregation pathway. However, so far no study of
amyloid protofibrils has been undertaken with high enough
resolution to allow the direct comparison of their morphology
with structural models of the amyloid protofilament. In the
current study we analyzed the morphological properties of TTR
amyloid protofibrils with atomic force microscopy (AFM) under
liquid buffer conditions. We show that protofibrils formed by mild
acidification display characteristics that are more closely related
to the double-helical model of the amyloid protofilament. These
structures are able to bind thioflavin T and Congo red and appear
to grow via an oligomer fusion mechanism. However, maturation
of the protofibrils into amyloid-like fibrils was not seen on a time
scale of several months. Rather, they showed a tendency to form
unorganized bundles that undergo dissociation at neutral pH.
Thus, the protofibrils described in our work possibly represent
a distinct structural entity whose potential cytotoxic activity—a
hallmark of oligomeric amyloid species—may be modulated by
environmental conditions that either preserve their protofibrillar
state, or induce their dissociation.
MATERIALS AND METHODS
Protein purification
Recombinant wild type (WT) TTR expressed in BL21 E. coli cells
was isolated and purified as described previously (Almeida et al.,
1997) followed by high-affinity anion exchange chromatography
(MonoQ column, GE Healthcare) equilibrated with 100 mM
BisTris pH 6.8. The protein eluting with approximately 150 mM
NaCl was subsequently concentrated and loaded onto a
calibrated analytical size exclusion chromatography superdex
S75 column (GE Healthcare) equilibrated with 50 mM HEPES pH
7.0 and 150 mM NaCl. Protein eluting in a peak corresponding to
tetrameric TTR (55 kDa) was collected and dialyzed overnight
against a weakly buffered solution (10 mM HEPES pH 7.0).
The protein was then concentrated to 10–15 mg/ml. Protein
concentration was determined with the Bradford method
(BioRad). Samples were approximately 98% pure as judged by
Coomassie-blue staining of SDS-PAGE gels. According to dynamic
light scattering measurements (Zetasizer Nano ZS, Malvern) the
samples were considered monodisperse. The hydrodynamic
radius of the particles ranged between 6.4 and 6.8 nm, consistent
with previous observations (Hou et al., 2007).
Sample preparation
TTR protofibrils were prepared by incubating WT TTR in 50 mM
sodium acetate buffer at pH 3.6, 378C and at a concentration of
1 mg/ml for a period of 1 year. At several time points following
acidification, samples were taken and diluted before surface
deposition to reduce molecular overcrowding. Thus, samples
were diluted 500-fold in acetate buffer at pH 3.6 and imaged
as described below. To test the stability of protofibrils at more
physiological conditions, samples in an advanced state of
aggregation were diluted 25-fold with phosphate-buffered saline
solution (PBS), followed by incubation at room temperature
for 1 min. Subsequently, the samples were diluted 20-fold in
ultrapure water (pH 6.3) prior to further investigation.
Atomic force microscopy
AFM imaging was carried out by using procedures reported in
previous papers on Aß fibrils (Karsai et al., 2005; Kellermayer et al.,
2005; Karsai et al., 2006; Karsai et al., 2007; Karsai et al., 2008;
Kellermayer et al., 2008) with modifications. Hundred microliters
of the diluted WT TTR sample was deposited on freshly cleaved
mica, incubated for 5 min at room temperature; the surface was
then rinsed with buffer. Imaging was carried out on a daily basis
over a period of 2 months and then periodically for up to 1 year.
AFM images of the samples were acquired with an MFP-3D AFM
instrument (Asylum Research, Santa Barbara, CA) in non-contact
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(AC) mode. Imaging was done by scanning in liquid, either in
50 mM acetate buffer, pH 3.6 or in ultrapure water for samples
which were incubated in PBS. Imaging was performed by using
low-noise cantilevers (Biolever, Lever A, Olympus) with a spring
constant of 30 pN/nm and a resonance frequency of 9.2 kHz.
The free amplitude was set to 0.3 V and the amplitude set point
to 0.2 V, and the images were recorded at a typical scanning
frequency of 0.8 Hz.
Image processing and analysis
Raw AFM images were flattened by using a linear function
followed by masking out particles with heights above 50 pm
and applying another first-order flattening to the (unmasked)
background to correct for artifacts arising from the first flattening
step. For any given image, a distribution of pixel heights was
used to find the background ‘height’ relative to which all height
measurements were carried out. Images were smoothed with
two passes of a Gaussian convolution filter with one pixel
neighborhood.
Protofibril length was measured by taking the tip convolution
effect into account (Figure 1). The cantilever tip was approximated by a hemisphere with a radius (R) of 30 nm (manufacturer’s
specifications) and the protofibrils as cylinders with diameter (h)
corresponding to the average protofibril height. Protofibril length
(L) was obtained from the measured fibril length (M) as:
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
L ¼ M2 2hRh2 :
(1)
For small oligomers that appeared nearly spherical under the
AFM, the molecular volume (V) was calculated by measuring the
height (h) and taking the full width at half height as the diameter
(d) which significantly compensates for the tip convolution effect
and these were used as follows (Carnally et al., 2008):
ph 2 3d 2
V¼
h þ
(2)
6
4
The number of TTR monomers present in a given oligomer
was estimated by considering the dimensions of the native WT
TTR monomer: 2 3 4.5 nm (Blake et al., 1978) or 27 nm3.
Statistical analysis of periodicity was carried out on several
protofibrillar axial sections which exhibited a clearly corrugated
structure. The data were analyzed in two independent ways.
In the first, the distance between consecutive local height
maxima or shoulders was measured, based on which a histogram
of periodicities was constructed. In the second, analytical approaches (autocorrelation and Fourier transformation) were
applied to axial height sections of 20 protofibrils taken from
high-resolution (1024 1024 pixel) images. The height values
were treated as a discrete homogeneous periodic signal by
ignoring the slant height profile at the protofibril termini and
considering only the harmonic component of the axial topology.
Thus, for any given harmonic segment of an axial section the
height variations (z) can be approximated as
zðdÞ ¼ h þ A sinð2p v d þ uÞ;
(3)
where h is the average height of the protofibril, A is the
height amplitude, v is the frequency, d is the distance along
the protofibril axis, and u is the phase. To obtain a statistical
representation of the major frequency components in protofibril
periodicity, an amplified waveform signal f(d) was obtained by
summing each i axial height section decremented by their
individual average height as
f ðdÞ ¼
n n
X
X
zi ðd Þhi ¼
ðAi sinð2p vi d þ uÞÞ;
i¼1
(4)
i¼1
where d 2 N0, 0 d 99. To allow amplification of the periodic
signal by limiting destructive phase interference, zi(0) was set at
u ¼ 908 which gives zi ð0Þ ¼ hi þ Ai . Since the periodic pattern is
expected to repeat itself over the analyzed protofibril length
(99 nm), a circular autocorrelation (CAC) was then obtained from
f(d) of protofibrils grown in pH 3.6 and from f(d) of protofibrils
obtained upon disassembly of bundles after PBS incubation.
To allow direct comparison between the two data sets a power
spectral density (PSD) was obtained from a normalized CAC
(nCAC). Thus, a discrete Fourier transform (DFT) of nCAC was
computed through a fast Fourier transform (FFT) algorithm with a
hanning window function and the data analyzed between
periods of 2–50 nm. All analyses were performed by using IGOR
Pro v6.06 (Wavemetrics, Oregon, USA).
Measurement of spectral properties
Thioflavin T Assay: 10 mM thioflavin-T (ThT) (e441 ¼ 2.2 104 M1 cm1) stock solution was prepared fresh in ThT buffer
(50 mM glycine/NaOH, pH 9.0). The solution was passed through
a 0.22 mm PVDF filter and stored in the dark and on ice. Native
WT TTR protein solution and protofibril suspension (1 mg/ml of
protein) were diluted 10-fold in ThT buffer. Emission was detected
at 482 nm with the excitation and emission slits set to 5 nm and
the spectrum recorded at room temperature on a Perkin-Elmer
LS50B fluorescence spectrometer.
Congo Red Redshift Assay: Measurements were carried out
as reported earlier (Bonifacio et al., 1996). Briefly, 5 ml of TTR
aggregates (1 mg/ml of protein) were mixed with 55 ml of a
10 mM Congo red solution prepared in 100 mM Tris/HCl, pH 7.0.
Spectra were recorded at room temperature in a Shimadzu
UV-2401 PC spectrophotometer.
RESULTS
Figure 1. Schematics of correction for AFM tip convolution in calculating the real length of TTR protofibrils.
High-resolution AFM investigation of WT TTR protofibrils was
carried out in the present work. Amyloidogenic transformation of
WT TTR was induced by acidification of the buffer solution. Within
the first week of incubation it was possible to observe monomers
and different size oligomeric structures (Figure 2A, inset) as
well as short flexible fibrillar structures of worm-like shape and
nodular morphology (Figure 2A). While monomers/dimers were
present throughout the entire incubation period (note the
background in Figures 2C and 3A), the number of oligomers
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R. H. PIRES ET AL.
Figure 2. Morphology and spectroscopic properties of WT TTR aggregates formed by incubation at pH 3.6. (A) WT TTR imaged by AFM on the 5th day
of incubation showing the presence of small protofibrils and oligomers (image 1024 512 pixels). Inset: magnified view of oligomeric structures (arrows)
with the indication of the estimated number of TTR monomers. (B) On the 6th day of incubation protofibrils appear longer, seem more frequent than
small oligomers, and already display some tendency to associate (image 1024 1024 pixels). Inset: magnified view of a protofibril displaying periodic
axial topographical substructure; the green line indicates trajectory along which the profile plot was obtained (see Figure 3D). (C) After 2 weeks of
incubation protofibrils show increased tendency to associate via their termini, forming a network containing nodules from which protofibrils extend in
quasi-radial symmetry as highlighted by circles (image 1024 2014 pixels). (D) 10 weeks of incubation lead to a formation of protofibril networks of
increased density forming protofibril bundles (image 512 512 pixels). (E) Fluorescence excitation spectra of Thioflavin T only (green), in the presence of
native WT TTR (blue), and in the presence of WT TTR protofibrils (red). A red shift is well observable in the latter case as expected for an amyloid aggregate.
(F) UV/VIS spectra of Congo red in the absence of protein (green), in the presence of native WT TTR (blue), and upon addition of protofibrils (red). The
presence of protofibrils induces a red shift in the absorbance spectrum that is characteristic of amyloid aggregates with a shoulder at 540 nm as seen from
the difference spectrum (black). Comparatively, the presence of native WT TTR does not induce any spectral changes as seen from difference spectrum
(gray).
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WILD-TYPE TTR PROTOFIBRIL STRUCTURE
Figure 3. Morphological properties of protofibrils before and after transient (1 min) incubation in PBS. (A) Protofibril bundle imaged 8 weeks after
sample incubation in 50 mM sodium acetate at pH 3.6. (B) Incubation for 1 min in PBS of the sample shown in (A) leads to the disassembly of bundles into
its constituting protofibrils. Inset: enlarged view of protofibrils displaying pronounced axial periodicity; the red line indicates trajectory along which
profile plot was obtained (see Figure 3D). (C) Contour length histogram of protofibrils in assembling conditions (pH 3.6, blue histogram) and following the
disassembly of bundles (PBS, red histogram). The arrowheads indicate local maxima spaced 15 nm apart for assembling protofibrils: 35, 50, 65, 80, 95 nm,
and spaced by 12 nm for protofibrils in disassembly conditions: 14, 26, 38 and 50 nm. (D) Topographical profile plots along the long axis of protofibrils in
assembling (blue, from Figure 2B) and disassembling (red, from Figure 3B) conditions; also indicated are the peak-to-peak distances in nanometers.
decreased while the fibrillar structures became increasingly
prevalent (Figure 2B).
We carried out a statistical analysis of the average topographical height of the filamentous structures. Because the observed
width is inflated by cantilever tip convolution, it is the height
data that was correlated with the thickness of the filaments. The
histogram of height data (Figure 4A) displayed normal distribution with a mean of 3.2 nm ( 0.8 nm SD, n ¼ 3505, 63
filaments), and a height variation amplitude of 1.0 nm (0.3 nm
SD, n ¼ 607, 127 filaments). Based on the thickness of the
filamentous structures, which is smaller than that of TTR amyloid
fibrils which are typically 10–12 nm (Serpell et al., 1995; Serpell
et al., 2000), we identify them as protofibrils and will refer to
them as such. Analysis of protofibril contour lengths revealed a
wide distribution between 30 and 300 nm (Figure 3C). In the
contour-length histogram local maxima can be observed, which
are separated by approximately 15 nm (arrowheads in Figure 3C).
The discrete distribution indicates that the length of the
protofibrils is an integer multiple of subunits with 15 nm
dimensions. Protofibrils may therefore be considered as a
linear assembly of 15-nm-long subunits. To further test for this
possibility we analyzed the height topography of the protofibrils
along the longitudinal axis. In the axial profile plot (Figure 3D,
blue curve) periodically spaced peaks and valleys can be
discerned. Statistical analysis of the peak-to-peak distance
(Figure 4B, blue histogram) shows an average distance of
15 nm (0.2 nm SD, n ¼ 266, 45 protofibrils). Thus, the protofibrils
indeed seem to be linear chains of subunits with axial dimensions
of 15 nm. Given their large dimensions compared with the
size of monomers, these aggregation subunits are likely to be
oligomers themselves.
Already within the first week, but extending throughout
the whole observation period, protofibrils showed a tendency to
associate into higher-order aggregates. An example of such an
aggregate, recorded in the second week of incubation, is shown
in Figure 2C. The figure displays an elongated structure with
approximately 650 nm in length that appears to result from
the association of several 100-nm-long protofibrils. Protofibrils
associate mostly via their termini, forming axial aggregates with
additional protofibrils annealing in a quasi-radial symmetry and
extending away from central nodules (Figure 2C, circles). This
type of architecture increased in size and complexity as a function
of incubation time, but it was not accompanied by an increase in
order. This is seen for example on an image obtained on the 10th
week of incubation where an arrangement that is more similar to
a protofibril bundle, rather than an amyloid fibril, can be seen
(Figure 2D). Although the sample was imaged for up to one year
after the start of incubation, no noteworthy changes where
observed henceforth; isolated protofibrils became scarcer and
only large bundles (1–4 mm2) were routinely present.
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R. H. PIRES ET AL.
Figure 4. Morphological analysis of protofibrils obtained on the pathways to assembly (acidic conditions, blue) and disassembly (upon incubation in
PBS, red). (A) Height distribution of protofibrils. (B) Histogram of periodicities obtained by measuring peak-to-peak distances. (C) Normalized
autocorrelation of statistically representative segments of protofibril cross-sections. (D) Power spectral density (PSD) of statistically representative
segments of protofibril cross-sections obtained by calculating the discrete Fourier transform (DFT) from the nCAC shown in Figure 4C.
To investigate whether the protofibril bundles display
canonical amyloid-like structure, we carried out spectroscopic
measurements to test for thioflavin-T (ThT) and Congo red
binding (Figure 2E and 2F). Protofibril bundles were positive for
ThT binding as observed by the increased fluorescence emission
(Figure 2E). The Congo red red-shift assay was also positive
(Figure 2F), as seen from the shoulder at 540 nm in the UV/vis
spectrum (red and black spectra), which is missing when only
Congo red is present (green spectrum) or when WT TTR was
added (blue and gray spectra). However, not only is the extent
of the shift much smaller than that obtained for ex-vivo fibrils
(Benditt et al., 1970), but the shift rapidly decayed on a the time
scale of a few minutes in the assay buffer, indicating instability of
the bundle structure at near-physiological pH. To test for possible
structural changes, we investigated the morphology of the
protofibril bundles with AFM following exposure of the sample to
more physiological conditions (PBS buffer) for 1 min as described
in the Materials and Methods section. The results are shown in
Figure 3, where a sample imaged on the 8th week of incubation
(Figure 3A) clearly exhibits a dense association of protofibrils
into a bundled structure. When the sample was subsequently
incubated briefly in PBS, the protofibril bundles disassembled,
protofibrils became more dispersed and showed a uniform
average thickness (Figure 3B). Analysis of protofibril contour
length following bundle disassembly again showed a distribution
containing several local maxima, but in this case the peaks were
separated by 12 nm (Figure 3C, arrowheads in red histogram).
Statistical analysis of the height distribution of these protofibrils
(Figure 4A, red histogram) indicates an average topographical
height of 4.5 nm (0.8 nm SD, n ¼ 3535, 50 protofibrils).
An enlarged view of the protofibrils resulting from bundle
disassembly (Figure 3B, inset) shows that, similarly to protofibrils
in the assembly conditions (pH 3.6), an axial topographical height
periodicity is present. However, the histogram of axial periodicity
of these protofibrils (Figure 4B, red histogram) shows a
much narrower distribution with a mean of 14 nm (0.01 nm
SD, n ¼ 133, 25 protofibrils). Although the difference in axial
periodicity between the assembly and disassembly conditions is
small, it is nevertheless significant according to statistical analysis
(unpaired Student’s t-test, p < 0.0001). Thus, protofibrils obtained
upon disassembly of bundles appear wider (by 1 nm) and with a
shorter periodicity (by 1 nm) suggesting that radial expansion
coupled with axial tightening of the protofibril structure has
taken place. In order to further evaluate if indeed an axial
compaction of protofibrils occurred upon bundle disassembly,
we carried out a detailed analysis of the axial topographical
periodicity along the protofibrils. After calculating a normalized
circular autocorrelation (nCAC) function of the axial topographical waveforms (Figure 4C) followed by discrete Fourier transformation (DFT) we obtained the power spectral density function
(Figure 4D) in which major periodicities were identified and the
relative amplitudes compared. Three major periodicities could
be identified: 28, 17, and 12 nm for assembling protofibrils
(blue curve), and 26, 13, and 8 for the disassembling ones (red
curve). These values are in close agreement with those obtained
from the histogram of periodicities and provide evidence
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WILD-TYPE TTR PROTOFIBRIL STRUCTURE
that upon disassembly of bundles the resulting protofibrils are
compacted and display reduced periodicities.
DISCUSSION
In the present work we investigated the assembly–disassembly
pathway of transthyretin amyloidogenesis with high-resolution
non-contact atomic-force microscopic imaging under liquid
conditions. The commitment of WT TTR towards the amyloidogenetic pathway was evoked by lowering the pH of the buffer
to 3.6. Acidification has been shown in the past to induce the
assembly of WT TTR into filamentous aggregates that exhibit
morphological, structural, and chemical properties similar to
those of tissue-formed amyloid fibrils (Bonifacio et al., 1996).
In recent years it has become clear that the aggregation reaction
in amyloidogenic systems is more complex than previously
thought, involving the assembly of oligomeric and protofibril
structures with very heterogeneous morphologies. The involvement of these species in amyloid fibril assembly is still debated
(Kodali and Wetzel, 2007). Because these pre-fibrillar species
appear to be the key agents in amyloid cytotoxicity, it is
important to characterize their structural properties in full detail.
Here we described the appearance of nodular protofibrillar
species in the amyloidogenic aggregation of TTR (Figure 2B
and 2C), with a topographical height that averaged 3.2 nm
and ranged between 2.7 and 3.7 nm (1 nm height amplitude)
(Figure 4A). The length repeat is consistent with the observed
topographical height periodicity (Figure 4B) and suggests that
protofibrils grow in stepwise fashion by the fusion of 15 nm
oligomers with the free end of the protofibril. Such an oligomer
fusion mechanism has been observed for other types of amyloid
protofibrils (Modler et al., 2003; Modler et al., 2004; Carrotta
et al., 2005; Hill et al., 2009). Similarly, we observe that the early
protofibrillar stages are characterized by the presence of small
oligomers (Figure 2A) that tend to disappear as protofibrils grow
in number. In this aggregation mechanism an oligomer is one
periodic unit. Interestingly, in an earlier work, the pre-fibrillar state
in the aggregation of WT TTR was reported to be populated by
8-nm globular oligomers (Cardoso et al., 2002) the size of which
is approximately one half of the periodicity observed here.
The 8 nm oligomers were found to disappear as 4–5 nm wide
protofibrils were formed, giving rise to higher order aggregates
of amyloid-like fibrils through hierarchical assembly (Cardoso
et al., 2002). We did not observe the formation of amyloid fibrils.
Instead, protofibrils appeared to bundle via their termini forming
clusters displaying quasi-radial symmetry rather than by lateral
association (Figure 2C). Protofibril bundles have been suggested
to form an intermediate state leading to the assembly of amyloid
fibrils (Arimon et al., 2005). This does not appear to be the case
with WT TTR under the conditions used in this study, where a
transition from protofibril bundles to mature fibrils was never
observed even after 1 year of study. The samples nevertheless
were positive for ThT (Figure 2E) and Congo red (Figure 2F),
which are classical amyloid markers. However, the spectral red-shift
of the Congo red absorption maximum decayed on the time-scale
of a few minutes, suggesting that disaggregation of the bundle
structure under conditions of the assay (neutral pH) may have
occurred. This observation is in accordance with several reports
that show that amyloid protofibrils and fibrils formed in acidic
medium can undergo depolymerization under neutral or alkaline
conditions (Yamaguchi et al., 2001; Yamamoto et al., 2005).
The possibility of instability of TTR amyloid protofibril bundles
at neutral pH conditions prompted us to undertake a morphological study. As seen from the AFM images (Figure 3A
and 3B), by incubation of protofibrils for 1 min in PBS resulted
in the disassembly of bundles into individual protofibrils. In
addition, small oligomers and monomers which populated the
background when imaging was performed at acidic buffer are no
longer present, suggesting that, since TTR is an acidic protein
with pI 5 (Connors et al., 1998), increasing the pH might affect
the protein surface charge to an extent that inhibits their
adsorption to the surface. The protofibrils thus obtained reveal
slightly altered structural features. The changes are characterized
by radial expansion, with the average height of protofibrils
increasing from 3.2 to 4.5 nm after PBS exposure (Figure 4A),
which can be partly explained by differences in electrostatic
interactions arising from the differences in pH and ionic strength
in the two samples (Muller and Engel, 1997). However, we also
observed that upon disassembly an axial compaction also
took place, as seen from the decrease in axial periodicity from 15
to 14 nm (Figure 4B). Although this difference is relatively small,
it is statistically significant ( p < 0.0001). In addition, while the
contour-length histogram of assembling protofibrils displays a
multimodal distribution with local maxima spaced at integer
multiples of 15 nm (Figure 3C, blue histogram), upon
disassembly of bundles each mode of the distribution becomes
separated by 12 nm (Figure 3C, red histogram). Both results
suggest that, upon disassembly a structural transition takes place
that induces a small but significant axial compaction of the
protofibrils. A similar observation can be made from the analysis
of the power spectral density (PSD) of the statistical representation of protofibrils (Figure 4D). The identified periodicities, 12, 17,
and 28 nm (on assembly) and 8, 13, and 26 nm (on disassembly),
are comparable to the values obtained from the statistical
measurement of peak-to-peak distances (Figure 4B). Since the
two types of analyses are fundamentally different is not possible
to directly compare the magnitude of the power in a PSD
curve with the magnitude in a periodicity histogram (counts/
frequency). However, the Fourier transform analysis of waveforms
is an extremely sensitive approach to study periodic structures,
allowing the identification of periods that could otherwise
be unnoticeable in a histogram derived from peak-to-peak
distances. While the histograms show a sensible shortening of
periodicities, these changes are far more noticeable in the PSD
analysis where periods identified on protofibril disassembly are
generally shorter than those in the assembly route (Figure 4D).
On the assembly and disassembly PSD curves, only a central
period at 12–13 nm appears to be a common feature, albeit with
different power. Upon disassembly, the 17 nm periodicity present
in protofibrils during assembly is no longer apparent, and this
change is accompanied by a noticeable increase in the power of
the 12–13 nm period. This shortening in periodicity by 4.5 nm is
within the dimensions of a single TTR monomer (Blake et al.,
1978). Thus, as suggested by the periodicity histogram, transient
incubation of protofibrils in PBS leads to a tightening of the
protofibril structure, a process that might be coupled to the
observed radial expansion. Additional periodicities were also
identified by the PSD analysis that are weakly represented in
the periodicity histogram, notably the 28 nm as well as the 8
and 26 nm periods found in assembling and disassembling
protofibrils, respectively. The shorter periods are likely to be
associated with very small height variations as indicated by their
smaller power in the PSD curves, and thus may have been
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R. H. PIRES ET AL.
occasionally overlooked during peak-to-peak distance measurements. On the other extreme are the large periods of 26
and 28 nm which are relatively large when compared to the
measured peak-to-peak distances, and may reflect a more global
morphological feature of the protofibril structure on binding to
the surface. Altogether, the morphological changes observed
likely stem from molecular rearrangements at the level of
individual monomers, underlying the structural dynamism that
is present within the TTR protofibril that could lead to the
disassembly of the protofibril bundles.
Since it has been proposed that protofibrils could be the direct
precursors of the protofilament, it is important to compare our
morphological data obtained here with the existing structural
models for the TTR amyloid protofilament. Analysis of electron
micrographs of ex vivo TTR amyloid fibril cross sections has revealed
that the protofilament is 4–5 nm in diameter (Serpell et al., 1995).
For the in vitro assembled TTR amyloid-like fibrils, protofibrils
measuring 4 nm were also found to be the fundamental polymeric
unit (Cardoso et al., 2002). In this latter work, mass-per-length
measurements in STEM data suggest that the unitary filamentous
structure is a linear array of monomers. This is in agreement with a
previous model for the TTR protofilament based on X-ray diffraction
data obtained on ex vivo fibrils (Inouye et al., 1998). In the report by
Cardoso and others, no periodic substructure was resolved for
protofibrils (Cardoso et al., 2002). However, molecular dynamics
simulations for a TTR protofilament modeled as a series of
monomers that form two extended b-sheets have proposed the
existence of a helical structure with a 48 b-strand repeat (Correia
et al., 2006), or a 23.04 nm periodic unit considering 0.48 nm as the
inter-strand distance. There is, however, another model for the TTR
protofilament also based on X-ray diffraction of ex vivo amyloid
fibrils, which is described as double-helical structure containing a
11.55 nm repeat and measuring 5 nm in height (Blake and Serpell,
1996; Sunde et al., 1997).
While the height of the protofibrils is in good agreement with
the diameter of the TTR protofilament, the periodicity of
12–13 nm detected on the PSD analysis is very close to the
11.55 nm repeat of the double-helical model, with a difference of
1 nm that is within the error of the measurements of our AFM
images in the XY-plane (1024 1024 pixels for 1 1 mm images).
Assuming an overall helical conformation for the protofibrils
described here, as it has been proposed for protofibrils from
lithostathine (Gregoire et al., 2001) the 15–17 nm periodicity
found in protofibrils in assembly conditions, may result from a
more relaxed conformation than that of the protofilaments which
are likely to have their structure more constrained by the
remaining fibril structure. In addition, transient incubation of the
same protofibrils in near-physiological conditions of pH and ionic
strength, lead to a structural transition where the height and
periodicities match quite remarkably the dimensions proposed
for the double-helical model of the protofilament.
Despite the morphological similarities between the TTR
protofibrils and the double-helical model of the protofilament,
the protofibrils did not assemble into higher order aggregates
that would resemble mature amyloid fibrils as seen from ex vivo
preparations. Thus, protofibrils, despite some morphological
similarity to current protofilament models, may have a very
distinct structural organization that effectively precludes their
hierarchical assembly into amyloid fibrils and may therefore be
considered ‘off-pathway’ products. However, it may also happen
that the protofibrils obtained here simply require an additional
factor that promotes their self-assembly into amyloid fibrils.
For example, ex vivo TTR amyloid fibrils are often found to contain
fragments of the protein that appear to influence fibrillar
morphology (Bergstrom et al., 2005). In addition, it is known that
the in vitro and in vivo assembly can be stimulated by a variety of
cellular components, including proteoglycans, glucosaminoglycans, lipids, or collagen (Relini et al., 2006; Relini et al., 2008; Naiki
and Nagai, 2009). Inclusion of these elements in future experiments
will yield further insights into the mechanisms of TTR amyloid
fibrillogenesis. Since the acidification of TTR induces the formation
of cytotoxic species before the pre-fibrillar state, it is very likely that
the protofibrils described here have a cytotoxic activity. The fact
that they manifest instability at physiological conditions is therefore
likely to be of relevance for the etiology of TTR-related amyloidoses.
CONCLUSIONS
We have described the morphological features of transthyretin
protofibrillar amyloid intermediates by using atomic force
microscopy under liquid conditions. The protofibrils, obtained
by mild acidification of the sample, display an axial periodicity
related possibly to the oligomeric fusion mechanism of their
assembly. The protofibrils aggregate into irregular bundles on a
time-scale of several weeks, but disperse into axially compacted
and radially expanded structures upon neutralizing the pH. The
periodicity observed here in protofibrils agrees with the
predictions of the double-helical model of TTR assembly. Despite
displaying the hallmarks of amyloid features, protofibrils failed to
aggregate into mature fibrils even during extended time periods.
Thus, the protofibrils seen here likely represent a kinetically
trapped structural variant, for which a precise structural
description awaits further high-resolution investigations. The
axial compaction and radial expansion indicate that the
protofibrils are nevertheless structurally dynamic, a fact that
may be relevant for their potentially cytotoxic activity.
Acknowledgements
The authors are thankful to András Kaposi for insightful comments and suggestions. This work was supported by grants
from the Hungarian Science Foundation (OTKA K73256), the
Hungarian National Office of Research and Technology
(NANOAMI KFKT-1– 2006–0021, OMFB-380/2006), and the Hungarian Medical Research Council (ETT-229/09) to MSZK; and
Project GRICES&FCT – Proj. 4.1.1-Hungary, Portugal and Project
n. 037525 EURAMY (FP6-LIFESCIHEALTH-6) from EU to AMD. RHP
acknowledges the award of a short-term travel grant by the
Calouste Gulbenkian Foundation of Portugal.
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