REVIEW ARTICLE
Mechanisms of amyloid fibril formation – focus on
domain-swapping
Eva Z
ˇ
erovnik
1
, Veronika Stoka
1
, Andreja Mirtic
ˇ
2
, Gregor Gunc
ˇ
ar
3
, Joz
ˇ
e Grdadolnik
2,4
,
Rosemary A. Staniforth
5
, Dus
ˇ
an Turk
1,6
and Vito Turk
1
1 Department of Biochemistry and Molecular and Structural Biology, Joz
ˇ

erovnik, Department of Biochemistry
and Molecular and Structural Biology, Joz
ˇ
ef
Stefan Institute, Jamova 39, 1000 Ljubljana,
Slovenia
Fax: + 386 1 477 3984
Tel: + 386 1 477 3753
E-mail: [email protected]
(Received 18 February 2011, revised 6 April
2011, accepted 28 April 2011)
doi:10.1111/j.1742-4658.2011.08149.x
Conformational diseases constitute a group of heterologous disorders in
which a constituent host protein undergoes changes in conformation, lead-
ing to aggregation and deposition. To understand the molecular mecha-
nisms of the process of amyloid fibril formation, numerous in vitro and
in vivo studies, including model and pathologically relevant proteins, have
been performed. Understanding the molecular details of these processes is
of major importance to understand neurodegenerative diseases and could
contribute to more effective therapies. Many models have been proposed
to describe the mechanism by which proteins undergo ordered aggregation
into amyloid fibrils. We classify these as: (a) templating and nucleation; (b)
linear, colloid-like assembly of spherical oligomers; and (c) domain-swap-
ping. In this review, we stress the role of domain-swapping and discuss the
role of proline switches.
Abbreviations
1D, 1 dimensional; AFM, atomic force microscopy; CO, critical oligomers; DA, dipole assembly; DCF, double-concerted fibrillation; IDPs,
intrinsically disordered proteins; MDC, monomer-directed conversion; NCC, nucleated conformational conversion; NDP, nucleation-
dependent polymerization; NP, nucleated polymerization; OFF, off-pathway folding; TA, templated assembly; TEM, transmission electron
microscopy; TFE, 2,2,2-trifluoroethanol.

cellular and animal models, and clinical studies. In
addition to providing a basic understanding of the pro-
cesses of protein folding and aggregation, such data
help towards translational approaches in medicine.
Structural and morphological data
Pre-amyloid, oligomeric intermediates, at the cross-
roads between protein folding and aggregation, possess
some common structure, regardless of their amino acid
sequence, because polyclonal antibodies raised against
one can bind to most such oligomers of different amy-
loid proteins [15]. It remains to be clarified whether
the structure of the prefibrillar oligomers is indeed all
b-sheet or whether the a-helical parts are the ones that
cross the membranes. As revealed by atomic force
microscopy (AFM), the structure of such annular olig-
omers embedded in lipid bilayers resembles that of the
well ordered bacterial toxins [15–17]. It still remains
for us to capture and image the annular oligomers in
their cellular environment where they are inserted in
cellular membranes. We envisage that two-photon fluo-
rescence correlation spectroscopy [18] may soon make
this possible. However, the common structural details
of the oligomers and their mode of toxic action remain
unknown [4] and would profit from innovative
research approaches.
Mature amyloid fibrils are long and straight, usually
comprising four to six filaments. They specifically bind
certain dyes such as Congo red and thioflavin T,
and they demonstrate a characteristic cross-b pattern
on X-ray diffraction, reflecting distances between

mers (or ‘globulomers’), ‘granules’, ‘critical oligomers’
or ‘spheres’. The a-pleated sheet structure would give
the globular oligomers higher dipole moments, which
would lead to a linear, colloid-like growth of amyloid
protofibrils. Glabe [28] suggested that, instead of
selecting oligomers by size, they could be selected by
the structural epitopes that become exposed. Trials
with conformationally selective antibodies have shown
that most of the prefibrillar species are bound by the
selective A11 antibody, and only a few by OC anti-
body, which also binds fibrils [28].
Comparison of amyloid aggregation
and protein folding
Under physiological conditions, protein folding takes
place in the crowded milieu of the cell with a whole
range of helper proteins [30]. These helpers include a
series of molecular chaperones whose functions,
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amongst others, are to prevent aggregation of incom-
pletely folded polypeptide chains [31] and to disaggre-
gate formed aggregates [32–34].
Protein folding involves a complex molecular recog-
nition phenomenon that depends on the cooperative
action of a large number of relatively weak, noncova-
lent interactions involving thousands of atoms. Hydro-
phobic [35,36], electrostatic [37–39] and van der Waals
interactions [40,41]; peptide hydrogen bonds [42,43];

stabilizes the b conformer, whereas the parallel orien-
tation of dipole moments destabilizes the a
R
conformer. However, the parallel arrangement of
dipole moments has advantages in polar solvents as a
result of favorable interactions with the solvent. There-
fore, the solvation of backbone atoms is much larger
for a conformers than for b conformers. Interaction
with solvent thus compensates for the destabilization
of the a conformation as a result of peptide dipole
moments. Alternation of the screening of backbone
electrostatic interactions by side chains causes different
conformational preferences of residues in aqueous
solution. Moreover, the additional modulation of
screening by changing the local environment and inter-
and ⁄ or intramolecular interactions may have a signifi-
cant influence on the preferential conformations of a
single amino acid residue. Therefore, even small varia-
tions in pH, temperature and ionic strength may have
sufficient potential to induce changes in the conforma-
tional propensities of amino acid residues to form sec-
ondary structure, as well as their ability to aggregate.
Computer simulations of protein aggregation indi-
cate that the hydrophobic effect plays an important
role in promoting the aggregation process [52]. Molec-
ular dynamics simulations of small peptides show that
b-sheet aggregates are stabilized by backbone hydro-
gen bonds, as well as by specific side-chain interac-
tions, such as hydrophobic stacking of polar side
chains and formation of salt bridges [53,54]. Coulom-

amyloid formation in vitro can be achieved by destabi-
lizing the native state of the protein under conditions
in which noncovalent interactions still remain favor-
able [65–67]. However, a local conformational change
before aggregation is not a necessary step in the fibril
formation of every protein. For some proteins, it was
shown that the native structure is preserved in the
fibrils [68,69]. Even all-a [70] or mixed a ⁄ b proteins
can transform into amyloid fibrils. It has also been
observed that the ability of a protein to undergo an
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a to b conformational change is facilitated by amino
acid regions that adopt an a-helical conformation
within the native structure, at the same time as having
a higher statistical propensity for the b-structure [71].
Mutations and changes in environmental conditions
both affect the aggregation reaction [72–76]. A protein
may assemble into amyloid fibrils with multiple distinct
morphologies in response to a change in amino acid
sequence [74] or upon a change in aggregation condi-
tions [23,24,76], as well as under the same growth con-
dition [22,77,78]. A study of b-lactoglobulin has shown
that charge repulsion makes amyloid fibrils more regu-
lar, whereas a lower charge, caused by a pH change in
the direction of the pI and⁄ or screening electrostatic
interactions by salt, results in shorter fibrillar rods that
pack into spheres [56].

fibril and (b) sufficient ‘conformational freedom’ of the
self-complementary segment to interact with other
molecules. Although self-complementary segments are
found in almost all proteins, the size of the amylome is
limited, suggesting that chaperoning effects have
evolved to prevent self-complementary segments from
interacting with each other [86].
Mechanisms of amyloid fibril formation
The models reported before the year 2000 have been
described in older reviews [63,64,87] and some excellent
reviews have been written subsequently [2,4,88–90]. On
the basis of the main features of the models, we have
classified them into three groups (Table 1): (a) templat-
ing and nucleation; (b) linear, colloid-like assembly of
spherical oligomers; and (c) domain-swapping.
For some of the case proteins relevant to the focus
of this review on domain-swapping, descriptions of
the mechanisms are provided, whereas, for most of the
other cases, the original publications are cited. On the
basis of our research on cystatins, which are capable
of domain-swapping, and on a literature survey of a
number of other amyloidogenic proteins that initially
form dimers, we emphasize domain-swapping as a pos-
sible mechanism underlying amyloid fibril formation
(see below). We also describe several factors that are
Table 1. Models for the mechanism of amyloid fibril formation.
Templating (A) and nucleation (B) Examples
a
A TA model [91] (Fig. 1A) Prion
A MDC model [92] (Fig. 1B) Prion, stefin B at pH 7

Stefin B at pH 5
(from dimers)
B Off-pathway model [137] with
domain-swapped oligomers
[121,122,163] and likely
propagated domain-swapping
Stefin A
a
All human proteins, with a representative case example.
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decisive for folding, misfolding, domain-swapping and
amyloid fibril formation.
Templating and nucleation models
Templating models comprise the templated assembly
(TA) and the monomer-directed conversion (MDC)
models. These models were originally proposed for the
prion protein transformations [91,92]. The TA and
DSC models are presented in Fig. 1A,B.
The ‘Polar zipper’ model proposed by Perutz et al.
[93] can also be classified as a templating model. This
model applies to amyloid forming proteins whose
b-sheets are stabilized by hydrogen bonds between
polar side chains, such as those between glutamine and
asparagine [94,95]. Molecular modeling has shown that
such polar residues link b-strands together into
b-sheets by a network of hydrogen bonds between the
main-chain amides and the polar side chains. The glu-

for both the nucleation and assembly rates. In this
model, a steady rate is ensured by an almost constant
concentration of the assembly competent oligomers
[98,103]. In the NCC model, the rate-determining step
is a conformational change that occurs in the nucleus
of preformed oligomers, rather than oligomer growth
itself. The concentration of soluble oligomers does not
increase with higher soluble protein concentration as a
result of the formation of assembly-ineligible com-
plexes. An example of NCC mechanism of amyloid
assembly is provided by the yeast prion protein Sup35
[103].
Linear colloid-like assembly of spherical
oligomers
Model of ‘critical oligomers’ (CO)
In the kinetics of yeast phosphoglycerate kinase fibril-
lation studied by Modler et al. [104], two steps were
observed during the formation of amyloid. ‘CO’ were
formed in the first step, whereas, in the second step, a
linear growth of oligomers into protofibrils was
observed. The kinetics of both steps were found to be
irreversible. Phosphoglycerate kinase was converted
into protofibrils, starting with a partially-unfolded
intermediate [105,106]. According to this model [104],
the acquisition of a b-sheet structure and fibril growth
are coupled events subsequent to a generalized diffu-
sion-collision process.
Dipole assembly (DA) model
Xu et al. [107] proposed a similar two-step model,
which they termed the ‘DA’ model. In the first step,

2
k
I
k
G
DimerMonomer Oligomer Fibril
Rearrangement
Protofibril
Off-path oligomer
A
B
C
D
E
F
G
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Domain-swapping as a mechanism of
amyloid-fibril formation
Here, we feel we need to explain more of our main
model proteins: cystatins and stefins. Given their
example, we illustrate the principle of domain-swap-
ping and how this can underlie the process of amyloid-
fibril formation.
Cystatins and stefins: an example of
domain-swapping proteins forming amyloids
Cystatins and stefins are a large family of cysteine pro-

(human cystatin C) [120] and by heteronuclear NMR
(human stefin A and chicken cystatin) [121,122]. The
domain-swapped dimer of stefin A (Fig. 2A) is made
of strand 1, the a-helix and strand 2 from one mono-
mer, and strands 3–5 from the other monomer
[120,122]. Similar to other cystatins, stefin B is prone
to form domain-swapped dimers (Fig. 2B). The 3D
structure of its tetramer [123] is composed of two
domain-swapped dimer units. The two domain-
swapped dimers interact through loop-swapping, also
termed ‘hand-shaking’ [123].
Folding mechanisms and oligomer formation by
domain-swapping
Folding studies are usually focused on unraveling the
conformational changes occurring within the mono-
meric protein under conditions often referred to as
‘physiological’, generally comprising pH 7.0 and room
temperature. It is clear that different folding conditions
must be examined when the focus switches to what is
occurring in the early steps of amyloid-fibril formation.
For many systems, including the stefins [124–126],
amyloid-fibrils form at nonphysiological pHs and in
the presence of further additives, such as metal ions or
Fig. 1. Schematic representations of the chosen mechanisms. (A) The TA model [98]. In the TA model, in a rapid pre-equilibrium step, the
soluble state (S) molecules that are initially in a random coil conformation bind to a pre-assembled (A) state nucleus. This binding induces
the rate-determining structural change from the random coil to the b-pleated sheet structure as the molecule is added to the growing end of
the fibril [91]. (B) The MDC model [98]. In the MDC model, a pre-existing monomer in the A-state conformation, analogous to the conforma-
tion adopted in the fibrils, binds to the soluble S-state monomer and converts it to an A-state dimer [92] in a rate-determining step. The
dimer then dissociates, and the constituent A-state monomers add to the growing end of the fibril. (C) The NDP model [88]. We consider
that the final structure labeled as ‘amyloid’ represents protofibrils rather than fibrils. The NDP model also predicts a lag phase that arises

lated, such as cystatin C and stefin B [128,129], are
more likely to form oligomers of the domain-swapped
type than those folding in a two-state (N-U) manner.
A number of conformational changes to the cystatin
molecule (as a representative of globular proteins)
undergoing oligomerization and, by extension, amyloid
formation will be considered below, including the role
of 3D domain-swapping and proline isomerization.
The energetics of domain-swapping
Intramolecular and intermolecular forces do not differ.
The only parameters favoring the monomeric state are
thus entropic. However, the edge strands usually pro-
tect a monomer from direct interaction with another
monomer [79], whereas the internal strands do not
possess such built-in protection. Under denaturing
conditions, the internal strands become exposed and
they can shift from intra- to intermolecular arrange-
ments. There also is considerable backbone strain in
the loop between strands 2 and 3 in the monomer
structure of stefin A [122] because this is required for
its proteinase inhibitory activity. The driving force for
dimerization may thus be the alleviation of this strain
as loop 1 extends on formation of the dimer [122].
Whether kinetic or thermodynamic factors govern the
oligomer formation remains to be clarified [130].
In certain proteins, metastable states can exist site
by site because the kinetic barriers are too high to
allow the energetic minimum to be reached in a rea-
sonable time [131]. However, when barriers are crossed
(e.g. by raising the temperature or pressure, by lower-

zation. Because native a-synuclein is not folded,
whereas stefin B is a globular protein, different interme-
diates may be rate-determining for fibrillation. Theoret-
ical studies [136] point to a role for hydrophobicity in
the nucleation barriers.
Fig. 2. Involvement of domain-swapping in amyloid fibril formation of cystatins. (A) Stefin A monomer (Protein Data Bank code: 1dvc) and
domain-swapped dimer as found in the structure of the tetramer (Protein Data Bank code: 1N9J); (B) stefin B monomer (Protein Data Bank
code: 1stf) and domain-swapped dimer (Protein Data Bank code: 2oct); and (C) proposed mechanism of the building up of amyloid fibrils
obtained on the basis of stefin B H ⁄ D exchange and heteronuclear NMR. Adapted from Morgan et al. [163].
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Thus, we have shown that domain-swapping of ste-
fins demands almost complete unfolding, with a high
activation energy of approximately 100 kcalÆmol
)1
pre-
ceding stefin A domain-swapped dimerization [121]. It
has been shown for RNAse A that dimerization is not
always energy demanding, as indicated by the presence
of a variety of different domain-swapped and non-
swapped dimers [130]. However, for stefins, a high
activation energy (as observed for domain-swapped
dimerization) is also a prerequisite for the initiation of
amyloid fibril growth [137] which, together with a
prominent role of the dimers accumulating in the lag
phase [126,127], supports the hypothesis that the
domain-swapped dimers are directly or indirectly
involved in the amyloid fibril formation of stefins. This

as preventing higher-order oligomerization. Further-
more, an excellent correlation between domain-swap-
ping and aggregation has been observed, which again
suggests a common mechanism.
In the structure of the monomeric stefin B in com-
plex with papain [142], the Pro103I is found to be
trans, whereas, in the tetrameric structure, the homolo-
gous residue Pro74 is cis. Hence, in the stefin B tetra-
mer, the proline residue in the loops undergoing the
exchange [123] has to isomerize from trans to cis.
Accordingly, in amyloid fibril formation of the wild-
type stefin B, the Pro74 cis isomeric state was found to
be critically important. Its mutation to Ser prolonged
the lag phase by up to ten-fold at room temperature
and almost stopped fibril growth [143]. Furthermore, it
was shown that the prolyl peptidyl cis–trans isomerase,
cyclophilin A, profoundly delayed the fibrillation rate
of the wild-type protein [143]. The potentially impor-
tant role of proline isomerization in stefin B oligomeri-
zation and fibril formation is also reflected in the
activation energy of approximately 27 kcalÆmol
)1
for
the fibril elongation phase [137], which is in the range
of proline isomerization reactions.
Pro32 is cis in the native structure of b
2
-microglobu-
lin. For this protein, cis to trans isomerism acts as the
‘gate-keeper’ for the transition to an intermediate con-

loop position differs in the tetramer from that in the
monomer and domain-swapped dimer. The monomer
and domain-swapped dimers of stefins A and B are
illustrated in Fig. 2.
Pro74 is widely conserved in stefins and cystatins,
and is found in trans isomeric state in all of the
reported structures [120,122,142,152,153]. Only in the
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high- resolution structure of the stefin B tetramer is it
in the cis isomeric state [123]. The dimer to tetramer
transition is associated with a rotation of domains,
which appears mandatory for the 90° repositioning of
the exchanged loops. From the superposition of stefin
B monomers and stefin A and cystatin C domain-
swapped dimers onto the tetramer structure, it is evi-
dent that the Ser72-Leu80 loops and the N-terminal
trunks have to adopt different conformation in the tet-
ramer to prevent clashes [154]. The adopted conforma-
tion of the Ser72-Leu80 loop and the N-terminal trunk
is made possible only by the proline in the cis confor-
mation.
Indirectly, we have confirmed that proline isomeriza-
tion is at the root of the slow conformational change
coupled to tetramerization by measuring the tempera-
ture dependence of the kinetics [123]. The value for the
activation energy of 28 ± 3 kcalÆmol
)1

[121,150]. In principle, any protein is capable of oligo-
merization by 3D domain-swapping [157]. Ogihara
et al. [158] designed a sequence of RNAse A that
underwent a reciprocated swap and another that ended
in a propagated swap (Table 1).
Under partially denaturing conditions, the protein
molecule partially opens and, when stabilizing condi-
tions are restored, the partially-unfolded monomers
can swap domains. When the exchange of secondary
structure elements is not reciprocated but propagated
along multiple polypeptide chains, this can result in
higher-order assemblies [159]. Guo and Eisenberg [160]
proposed the term ‘run-away domain-swapping’ mech-
anism for such a process of continuous domain-swap-
ping.
In their study of T7 endonuclease, Guo and Eisen-
berg [160] define ‘run-away domain-swapping’ as a
mechanism in which each protein molecule swaps a
domain into the neighboring molecule along the grow-
ing fibril. By designing disulfide bonds that form only
at the domain-swapped dimer interface, they were able
to show that the resulting covalently-linked fibrils con-
tained domain-swapped dimers. If these were locked in
a close-ended dimeric form by making internal disul-
fide bonds, they were unable to form fibrils. A study
by Liu et al. [161] indicates that the b-sheet spine in
amyloid fibrils of b
2
-microglobulin could be made
from amyloidogenic peptide sequences of the hinge

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An example of the propagated domain-swapping is
provided by human cystatin C. Amyloid fibril forma-
tion by human cystatin C has been studied [162,164]
and connected to the domain-swapping of this mole-
cule [120,165]. In an experiment where dimer forma-
tion was prevented by engineered disulfide bridges,
fibril formation was also prevented [165]. From these
studies, it is apparent that dimers play an important
role in fibril formation, although this does not imply
they should build the fibrils directly.
A further example of a domain-swapping mechanism
underlying amyloid fibril formation is provided by the
human stefins. In vitro studies have demonstrated that
stefin A, albeit under rather harsh preconditioning, is
able to form amyloid fibrils [64,125], as would be
expected if this process is generic [66]. In stefin A
monomer and dimer, there are more salt bridges inter-
connecting the a-helix and strand 2 to the rest of the
structure than in its stefin B homolog [59,125]. Conse-
quently, stefin A can only form amyloid fibrils in vitro
under very stringent conditions compared to the
almost physiological conditions needed for stefin B
[64,166]. Fibrillation of stefin A can be initiated by
heating the protein to predenaturing temperatures of
approximately 90 °C, which promotes domain-
swapped dimer formation, and by reducing the pH
below 2.5, which partially unwinds the dimer [121].

their lifetime increases when they become more abun-
dant [137,168].
An example of the OFF model, involving domain-
swapping, is provided by human stefin B (Fig. 1G).
The fibrillation of stefin B at room temperature and at
approximately pH 5 is characterized by an extensive
lag phase, in which granular aggregates have been
observed by TEM and AFM, appearing as micelle-like
arrangements of oligomers [124–127,169]. After the lag
phase, various morphologies have been detected during
the fibril growth phase, from annular to spherical, rod-
like and amorphous species [126,169]. Unlike at room
temperature, at temperatures above 35 °C, thioflavin T
fluorescence shows no visible lag phase [137]. The sub-
sequent growth phase shows an anomalous dependence
on protein concentration; at low concentrations, the
final value is reached faster than at higher concentra-
tions. This observation is explained in terms of an off-
pathway state with a rate-limiting escape rate [137].
However, there may be two (or more) pathways by
which this protein aggregates, depending on pH and
ionic strength [124,126,127].
Discussion
Although it is essential to study different conforma-
tional states populated by the amyloid precursor pro-
teins, it is a difficult task to draw links between states
occurring during folding and the ‘misfolded’ states
populated during amyloid formation. In some cases,
extensive study allows us to determine the pathways to
which different conformations belong. However, our

is therefore not simply to determine the conditions that
are sufficiciently destabilizing to favor large conforma-
tional changes but, instead, those that are sufficiciently
stabilizing to produce a new structure. It is tempting
to imagine that these changes are controlled by nature,
just as they can be by the scientist.
In the case of globular proteins, the formation of
partially-unfolded intermediates populated from the
native state or accessible during refolding is the first
critical step of the pathway of amyloid fibril formation
[61,170,171]. Taking the example of stefin B, partial
unfolding is a prerequisite for both protofibril and
fibril formation. We have observed that protofibrils
tend to form from the structured molten globule
obtained at pH 3.3 and the mature fibrils from the
partially-unfolded monomer (native-like intermediate)
populated at pH 4.8, which transforms into domain-
swapped dimer [124,126,127]. We also have shown that
the aggregates formed from different partially-folded
intermediates differ in toxicity [172].
The intrinsically disordered proteins (IDPs) [173],
constitute a large fraction of naturally occurring amy-
loidogenic proteins [174]. In the case of IDPs (i.e.
natively unfolded proteins), the formation of partially
structured conformers occurs by partial folding, and
fibril formation is promoted by factors that induce
partial folding [12,13,175]. For example, in the case
of a-synuclein, either a decrease in pH or an increase
in temperature appears to induce partial folding, as
well as enhance the propensity of the protein to fibril-

dimers [126,127], and possibly tetramers [155], cannot
form. Therefore, partially-unfolded monomers and
dimers accumulate until they form the so-called ‘criti-
cal oligomers’, which comprise part of the insoluble
granular aggregate. When such a critical mass is
reached and the oligomeric spheres gain sufficiently
large dipole moments, they form linear chains in the
form of colloid particles to give protofibrils. These can
interact laterally, building up fibrils. Under suitable
solvent conditions, the protofibrils smooth out into fil-
aments, which wind around each other and form
mature fibrils, whereas, under some other solvent con-
ditions, they remain protofibrillar. It is also possible
that several fibril morphologies could exist side by
side.
In the NP, NDP and NCC models (Table 1), the
partially-unfolded intermediates, when present at a
critical concentration, slowly assemble into a nucleus,
within which the first conformational change takes
place. These oligomeric nuclei then rapidly grow into
globular oligomers, also termed ‘granules’ or ‘spher-
oids’ and, after reaching a ‘critical’ size, go on to form
chain-like protofibrils (CO and DA models), which
eventually form fibrils [97,176] or remain protofibrillar.
Some other models, such as the DCF model, predict
that a second concerted conformational change has to
take place within the globular oligomers, after which
they can chain up (i.e. as colloid particles) into
protofibrils [102,104].
We have noted that the amyloid fibril formation of

folding on one end and noncooperative transitions via
multiple intermediates on the other, with all the rest
inbetween? [180]. In folding, the energy landscape rep-
resentation [181] is used to show different scenarios,
with steep funnels or ragged surfaces, slowly descend-
ing into a final funnel. In certain cases of metastable
states, the funnels can end in two or three minima.
We propose that such metastable states preceding
fibril formation could well be domain-swapped dimers
and higher oligomers, preceding or gate-keeping the
amyloid fibril formation. High-energy barriers of the
order of 100 and 30 kcalÆmol
)1
occur in the domain-
swapped dimerization of stefin A [121] and in the tet-
ramerization of stefin B [123], respectively. These barri-
ers are equivalent to those corresponding to almost
complete unfolding and proline isomerization. Both
barriers occur in amyloid-fibril formation by this pro-
tein [137]. We further propose that the term ‘propa-
gated domain-swapping’ would encompass both
domain-swapping and loop-swapping. The domain-
swapping demands almost complete unfolding
(> 90 kcalÆmol
)1
) and loop-swapping, usually an
extensive conformational change involving proline cis–
trans isomerization. One such reaction would cost
28 kcalÆmol
)1

We thank Professor R. H. Pain for editing the English
and making useful suggestions.
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