Stability and fibril formation properties of human and
fish transthyretin, and of the Escherichia coli
transthyretin-related protein
Erik Lundberg
1
, Anders Olofsson
2
, Gunilla T. Westermark
3
and A. Elisabeth Sauer-Eriksson
1
1 Department of Chemistry, Umea
˚
University, Sweden
2 Department of Medical Biochemistry and Biophysics, Umea
˚
University, Sweden
3 Division of Cell Biology, Diabetes Research Centre, Linko
¨
ping University, Sweden
Transthyretin (TTR) is a homotetrameric plasma pro-
tein that binds and transports the thyroid hormones
3,5,3¢-triiodo-l-thyronine and 3,5,3¢,5¢-tetraiodo-l-thyr-
onine (thyroxine) and retinol by binding to the retinol-
binding protein when it is loaded with retinol [1]. TTR
is mainly expressed in the adult liver, the choroid
plexus of the brain, and the retina [2,3]. TTR is
involved in three amyloid diseases: familial amyloidotic
polyneuropathy, familial amyloidotic cardiomyopathy
(FAC), and senile systemic amyloidosis (SSA) [4,5].
Whereas SSA is associated with native TTR, point

estingly, different structures displayed different tinctorial properties. hTTR
and sbTTR formed thin, curved fibrils at low pH (pH 2–3) that bound
thioflavin-T (thioflavin-T-positive) but did not stain with Congo Red (CR)
(CR-negative). Aggregates formed at the slightly higher pH of 4.0–5.5 had
different morphology, displaying predominantly amorphous structures.
CR-positive material of hTTR was found in this material, in agreement
with previous results. ecTRP remained soluble at pH 2–12 at ambient tem-
peratures. By raising of the temperature, fibril formation could be induced
at neutral pH in all three proteins. Most of these temperature-induced
fibrils were thicker and straighter than the in vitro fibrils seen at low pH.
In other words, the temperature-induced fibrils were more similar to fibrils
seen in vivo. The melting temperature of ecTRP was 66.7 °C. This is
approximately 30 °C lower than the melting temperatures of sbTTR
and hTTR. Information from the crystal structures was used to identify
possible explanations for the reduced thermostability of ecTRP.
Abbreviations
AFM, atomic force microscopy; BME, b-mercaptoethanol; CR, Congo Red; DSC, differential scanning calorimetry; ecTRP, Escherichia coli
transthyretin-related protein; EM, electron microscopy; FAC, familial amyloidotic cardiomyopathy; hTTR, human transthyretin; rTTR, rat
transthyretin; sbTTR, sea bream transthyretin; SSA, senile systemic amyloidosis; ThT, thioflavin-T; TLP, transthyretin-like protein; TRP,
transthyretin-related protein; TTR, transthyretin.
FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS 1999
diversity in age of onset, penetrance, and tissues affected
[7,8]. SSA is a geriatric disease affecting approximately
25% of the European Caucasian population over
80 years of age [4]. Like FAC, SSA is characterized by
heavy deposits of amyloid fibrils in the heart.
Structures of TTRs from different species have been
studied [9], including human transthyretin (hTTR)
[10–12], rat TTR (rTTR) [13], chicken TTR [14], and sea
bream TTR (sbTTR) [15,16]. Within the TTR family,

To understand the mechanism behind hTTR dissoci-
ation, misfolding, and amyloid formation, studies from
other species have provided valuable information. Like
hTTR, rTTR forms amyloid-like fibrils in vitro after
partial acid denaturation [32]. rTTR shares 85%
sequence identity with hTTR, which raises the question
of how important tertiary similarities are, as opposed
to sequence identity, for the ability of the protein to
form fibrils. In an attempt to answer this question, we
have investigated the fibril-forming properties of
sbTTR and ecTRP in vitro, and compared the results
with those of hTTR.
Our results showed that hTTR, sbTTR and ecTRP
can form fibrillar structures, but under different solu-
tion and temperature conditions. Furthermore,
depending on the conditions used, fibrils of different
morphology were obtained. Recent studies have shown
that sbTTR binds thioflavin-T (ThT) at low pH, sug-
gestive of amyloid [33]. In our study, we verified that
sbTTR forms fibrillar structures at low pH that are
similar in shape to those of hTTR. We also found
that, even though  70% of the amino acids of ecTRP
are different from the respective amino acids in hTTR
and sbTTR, ecTRP has the ability to form Congo Red
(CR)-positive fibrils in vitro if the temperature is
increased sufficiently. Similar findings for ecTRP were
published while this work was in progress [34]. The
Fig. 1. Multiple sequence alignment of representatives of TRPs, TTRs, and TLPs. There are 57 gene clusters in Caenorhabditis elegans,
referred to as TTR-1 to TTR-57 in
WORMBASE. Some of these sequences were originally identified as being structurally TTR-like by Sonnham-

TTR G. gallus
TTR H. sapiens
TTR S. aurata
TRP E. coli
TRP C. elegans R09H10.3
TRP C. elegans ZK697.8
TRP M. musculus
TRP B. subtilis
TLP X. index
TLP R. similis
E. Lundberg et al. Stability and fibril formation of TTR and TRP
FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS 2001
results emphasize the potential for amyloid formation
as a common property of all proteins, a feature that
can sometimes even bring new functionality [35–37].
The thermal stability of ecTRP was found by differen-
tial scanning calorimetry (DSC) to be approximately
30 °C lower than that of sbTTR and hTTR. Compara-
tive studies of structures from homologous thermophiles
and mesophiles have revealed several factors that gener-
ally contribute to the intrinsic thermal stability of pro-
teins [38–40]. These include tighter hydrophobic packing
of the protein core [41–43], increased electrostatic inter-
actions on the surface of the protein [44,45], more pro-
lines and alanines [46,47], and increased hydrogen
bonding of the polypeptide chain [48–50]. In addition,
improved intersubunit contacts within oligomeric pro-
teins contribute to protein stability [51,52]. Here, we
analyze the structural basis for the differences in ther-
mostability of hTTR, sbTTR, and ecTRP.

protofilaments [54]. At the pH interval 4.0–5.5, predomi-
nantly amorphous aggregates, rather than fibrillar struc-
tures, were observed in the hTTR samples (Fig. 3A). It
is, however, not possible to quantitatively estimate the
ratio of aggregates to fibrillar structures from the AFM
images. The hTTR and sbTTR samples were also visual-
ized with electron microscopy (EM). The EM images
were consistent with the morphologies that we eluci-
dated from the AFM images, and verified that fibrillar
structures were present in the protein samples at pH 4.5
(Fig. 3B). To determine whether the lack of fibrillar
structures in the hTTR and sbTTR samples at pH 4.5
could be an effect of the technique used for analysis
(that is, the fibrils are unable to bind to mica gels at this
pH), fibril-containing samples of hTTR formed at
pH 2.0 were adjusted to pH 4.5 and incubated for vari-
ous lengths of time. AFM images of this material
showed that the fibrils formed at pH 2.0 persisted at
pH 4.5, thereby verifying that these fibrillar forms can
bind to mica gels even at higher pH (data not shown).
Tinctorial properties of fibrillar structures formed
at low pH
The protein fibrils and aggregates obtained by the
partial acid denaturation experiments were tested for
ThT, which is a fluorescent dye commonly used to
Fig. 2. Turbidity assays for hTTR, sbTTR,
and ecTRP. The turbidity was measured at
330 nm after incubation of protein samples
at 37 °C for 72 h.
Stability and fibril formation of TTR and TRP E. Lundberg et al.

B
C
ab
ab
Fig. 3. (A) AFM images of hTTR (left) and sbTTR (right). The samples were incubated at 37 °C for 72 h. Fibrils were present in samples incu-
bated at pH 2.0–3.5, whereas aggregates were predominantly present in samples incubated at pH 4.5–5.5. No fibrils or aggregates were
detected in the ecTRP samples, at any of the pH intervals tested (pH 2.0–12.0; data not shown). The white scale bar is 500 nm. (B) EM
images of fibrils of hTTR (a) and sbTTR (b) incubated at pH 4.5. (C) CR staining of hTTR incubated for 3 days at pH 4.5. (a) shows fluores-
cence (at 594 nm) and (b) shows the characteristic apple-green birefringence with polarized light.
E. Lundberg et al. Stability and fibril formation of TTR and TRP
FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS 2003
any pH range. Also, the large amounts of small fibril-
lar hTTR and sbTTR structures, observed with AFM
(Fig. 3A) at pH 2.0–3.0, did not stain with CR.
Apparently, ThT is more promiscuous than CR in
binding to thinner immature fibrillar structures of
TTR.
To determine whether the thin and curved protofi-
brils formed at pH 2.0 by hTTR and sbTTR could be
induced to form amorphous aggregates or thicker fila-
ments, the samples were readjusted to a pH of 4.5.
Interestingly, even after 6 weeks at 8 °C, these samples
contained the same type of protofibrils. This implies
that the low-pH-induced and ThT-binding fibrils are
not readily converted to CR-positive fibrillar aggre-
gates or thicker filaments, or at least not under these
conditions.
Fiber formation induced by heating
Fibrillar structures of hTTR, sbTTR and ecTRP were
obtained by heating the protein samples for 72 h with-

seems to be the same as for the fibers seen at lower pH. ecTRP
does not bind ThT at any pH level.
A
B
hTTR
sbTTR ecTRP
0.2 µm
Fig. 5. (A) AFM images of hTTR, sbTTR and
ecTRP heated at 55 °C (hTTR) or 65 °C
(sbTTR and ecTRP), respectively, for 72 h.
The fibril heights were estimated to be
 2.8 nm for hTTR,  3.5 nm for sbTTR,
and  4.0 nm for ecTRP. The white scale
bar is 500 nm. (B) Left: EM image of ecTRP
from the same sample as in (A). The mate-
rial shows fluorescence (at 594 nm) (middle)
and apple-green birefringence (right) after
visualization with polarized light.
Stability and fibril formation of TTR and TRP E. Lundberg et al.
2004 FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS
values lower than 5.0, as shown previously [53], sbTTR
and ecTRP did not completely separate into their
monomeric units until the pH values were below 4.6
and 3.0, respectively (Fig. 6). In agreement with the
partial acid denaturation experiments, the ecTRP tetra-
meric structure seems to be more resistant to varia-
tions of pH. However, the SDS ⁄ b-ME treatment
generally dissociated a larger fraction of the ecTRP
protein material than of hTTR and sbTTR into mono-
meric structures. This behavior is likely to reflect

value approx-
imately 26 °C below the values for both sbTTR and
hTTR (Fig. 7). The inability of ecTRP to form fibrils
at low pH can therefore not be directly correlated with
the thermostability of its tetrameric and monomeric
structures.
Thermostability and protein structures
We have analyzed the structures of hTTR (Protein
Data Bank code: 1F41 [12]), sbTTR (Protein Data
Bank code: 1SN2, [16]) and ecTRP (Protein Data
Bank code: 2G2N [24]) in an attempt to identify
factors that contribute to their profound differences in
stability. The results are summarized in Table 1.
Introduction of alanines, prolines and aromatic resi-
dues can contribute to protein stability [46,47,52].
hTTR and sbTTR have more alanine residues than
ecTRP, 12 versus eight and 13 versus eight, respec-
tively, which might contribute to entropic stabilization.
On the other hand, ecTRP has more aromatic residues
than hTTR and sbTTR, 13 versus 12 and 13 versus
Fig. 6. Analysis of tetramer stability by SDS ⁄ PAGE. The samples
were not boiled. The tetrameric structures of sbTTR and ecTRP
show increased stability at lower pH as compared to hTTR. Nota-
bly, both hTTR and sbTTR are unaffected by the SDS ⁄ BME treat-
ment, and remain either in a monomeric or a tetrameric state,
depending on the pH of the protein buffer. In contrast, SDS ⁄ b-ME
treatment alone dissociates a fraction of the tetrameric ecTRP into
the monomeric state at all pH values.
Fig. 7. DSC profiles of hTTR, sbTTR, and ecTRP. The melting
temperatures (T

The protein volumes of single subunits and tetra-
meric structures for the three proteins were investi-
gated. Our calculations show that there is a decrease in
the protein volume of each monomer that is related to
an increase in thermal stability. Furthermore, there is
a clear correlation between thermostability and molec-
ular volume occupied by the tetramers (Table 1). The
difference between the tetrameric volume and the
volume of the corresponding number of monomeric
units, DV, is negative in all cases, demonstrating that
the protein density increases slightly upon tetrameriza-
tion.
Analysis of the number of hydrogen bonds formed
within monomers and dimers revealed pronounced dif-
ferences between the three proteins. As previously
mentioned, the hTTR and sbTTR structures contain
buried polar residues and water molecules. These
waters allow the formation of 10 more hydrogen bonds
within their monomeric units, and about 20 more
hydrogen bonds within their dimeric units, than in the
ecTRP protein structure (Table 1). In addition, 14 and
15 hydrogen bonds are formed at the monomer–mono-
mer interface of sbTTR and hTTR, whereas only eight
are formed in ecTRP. Five hydrogen bonds are formed
across the dimer–dimer interface of both hTTR and
sbTRP, whereas three are formed in ecTRP (Table 1).
Discussion
TTRs and TRPs are two protein families with similar
structures but different functions, due to divergent
evolution. The TTRs, found only in vertebrates, func-

Glu, Asp 17 14 12
Lys, Arg 12 10 13
Salt bridges
a
553
Protein volume (A
˚
3
)
b
Monomer, V
m
11 490 11 340 11 810
Tetramer, V
t
45 760 45 190 47 100
DV = V
t
) 4V
m
)200 )170 )140
Hydrogen bonds
c
Monomer 75 (12) 70 (11) 64 (4)
Dimer 165 (20) 163 (20) 141 (10)
a
A distance less than or equal to 4 A
˚
between charged groups
defines an ion pair [74].

acid and thermal denaturation. Some differences were
apparent. Analysis with SDS ⁄ PAGE showed that hTTR
is less stable than the other proteins under acidic condi-
tions, and dissociates into monomers when the pH falls
below 5.0. sbTTR shows similar behavior, and is only
marginally less sensitive to acidic pH, dissociating at a
pH below 4.5. Interestingly, we found that ecTRP main-
tains its tetrameric structure even at very low pH values.
Different results were obtained when the protein
samples were analyzed with DSC. The melting point for
ecTRP was determined to be 66.7 °C, which is more
than 26 °C lower than those of both hTTR and sbTTR
(Fig. 7). Therefore, whereas previous SDS ⁄ PAGE
analysis suggested that the tetrameric structure of
ecTRP is more stable than that of hTTR [34], the DSC
results showed that ecTRP is significantly less thermo-
stable than either hTTR or sbTTR.
Comparison of the crystal structures of hTTR,
sbTTR and ecTRP highlights a number of structural
differences that are consistent with the current explana-
tions of thermal stability in proteins. Noteworthy is the
reduced number of negatively charged residues in the
ecTRP structure. This could possibly also explain its
structural stability at low pH. Furthermore, the ther-
mostable TTR proteins have more hydrogen bonds and
ion pairs, and their structures are more densely packed
than that of ecTRP. Thus, it seems that the differences
in thermostability are mainly due to the presence of
specific polar and charged residues in hTTR and
sbTTR, which form additional hydrogen bonds that

formation. Generally, where fibrils were observed for
hTTR, fibrils of similar morphology were observed
also for sbTTR, after some minor adjustments of the
fibrillization protocol. We did not detect any CR-posi-
tive fibrils of sbTTR at pH 4.5–5.5. This suggests that
hTTR forms amyloid fibrils by partial acid dissociation
more readily than sbTTR. The result does not exclude
the possibility that sbTTR can form CR-positive fibrils
at low pH, but more samples need to be examined, or
the concentration of protein needs to be increased.
ecTRP shares 30% sequence identity with hTTR. In
agreement with previous reports [34], we detected
CR-positive fibrils of ecTRP induced by heating. These
fibrils are similar both in shape and in dimension to
the fibrils of hTTR and sbTTR formed by heating.
The thick and straight morphology of heat-induced
fibrils of hTTR, sbTTR and ecTRP is similar to that
of amyloid fibrils in vivo. We have so far not been able
to convert thin and ThT-positive protofibrils of hTTR
and sbTTR, formed at low pH, to thicker and
CR-positive structures, suggesting that the kinetics are
very slow. This suggests that the TTR amyloid archi-
tecture is not the result of only one highly stringent
assembly of structures.
In the past, the propensity of proteins to form fibrillar
structures has most often been associated with disease.
Recently, however, examples have been presented where
conformational changes and fibril formation are
associated with an advantageous gain of function [37]. It
is not clear whether the fibril formation properties of the

the cells were grown overnight at 30 °C.
Similar purification protocols were used for hTTR and
sbTTR. Frozen cells were thawed in 20 mm Tris ⁄ HCl
(pH 8.0) and 50 mm NaCl, and lysed by sonication in the
presence of DNase I. Cell debris was removed by ultra-
centrifugation (120 000 g for 40 min) at 4 °C. The lysate was
filtered through a 0.2 lm syringe filter (Millipore Corpora-
tion, Bedford, MA, USA), and purified on a Q-Sepharose
Fast Flow anion exchange column (GE Healthcare,
Uppsala, Sweden) equilibrated with 20 mm Tris ⁄ HCl
(pH 8.0) and 50 mm NaCl, and eluted with a linear gradient
(0.1–1 m NaCl in 20 mm Tris ⁄ HCl, pH 8.0). TTR fractions
were pooled and concentrated (Centriprep-10; Amicon
Inc., Beverly, MA, USA), and then further purified by gel fil-
tration on a HiLoad 16 ⁄ 60 Superdex-75 (GE Healthcare)
column with buffer containing 20 mm Tris ⁄ HCl (pH 6.8)
and 50 mm NaCl. Pure TTR fractions were pooled, concen-
trated to 5 mgÆmL
)1
(Centriprep-3; Amicon), and stored at
)20 °C. ecTRP was cloned, expressed and purified as
previously described [19], using 50 mm Tris ⁄ HCl (pH 7.0)
and 200 mm NaCl as buffer in the final gel filtration step.
The pure ecTRP fractions were pooled and stored at )20 °C.
Partial acid denaturation
Denaturation studies were performed according to a previ-
ously described protocol for hTTR [53]. hTTR, sbTTR and
ecTRP were dialyzed against 2 mm NaH
2
PO

water, and air dried. The surface was analyzed with
a Nanoscope IIIa multimode atomic force microscope
(Digital Instruments, Santa Barbara, CA, USA), using
Tapping Mode in air. A silicone probe was oscillated at
around 270 kHz, and images were collected at an opti-
mized scan rate corresponding to 1–4 Hz. The images were
flattened and presented in height mode using nanoscope
software (Digital Instruments).
EM
Negative staining for EM was performed on the same sam-
ples used in the AFM studies. For this purpose, the material
was centrifuged at 16 000 g for 30 min, after which most of
the supernatant was removed and 200 lL of distilled water
was added. The material was vortexed, and aliquots of
3–5 lL were applied to Formvar-coated copper grids.
Contrast was achieved with 2% uranyl acetate in 50% etha-
nol, and the material was studied at 100 kV in a Jeol 1230
electron microscope (Jeol, Akishima, Tokyo, Japan).
CR-binding studies
For analysis with CR, 1–2 lL of diluted, vortexed samples
were applied to microscope slides and air dried. CR stain-
ing was performed according to Puchtler et al. [71], and
examined by light microscopy. The presence of amyloid
was verified by the green birefringence in polarized light
and with red fluorescence in a microscope equipped with
filters for wavelengths at 560 nm (excitation) and 590 nm
(emission).
ThT-binding studies
Protein samples incubated at 37 °C for 72 h were vortexed,
and 25 lL aliquots were mixed with 173 lL of a buffer

was
used as a control for these experiments.
Acknowledgements
The authors wish to thank Uwe H. Sauer and Tobias
Hainzl for valuable discussions, and Terese Bergfors
for critical reading of the manuscript. This work was
supported by grants from the Swedish Research Cou-
ncil, the FAMY ⁄ AMYL patients’ association, the
Kempe Foundation, and the Gustafsson Foundation.
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