REVIEW ARTICLE
Amyloid-fibril formation
Proposed mechanisms and relevance to conformational disease
Eva Z
ˇ
erovnik
Department of Biochemistry and Molecular Biology, Jozˇef Stefan Institute, Ljubljana, Slovenia
The phenomenon of the transformation of proteins into
amyloid-fibrils is of interest, firstly, because it is closely
connected to the so-called conformational diseases, many of
which are hitherto incurable, and secondly, because it
remains to be explained in physical terms (energetically and
structurally). The process leads to fibrous aggregates in the
form of extracellular amyloid plaques, neuro-fibrillary
tangles and other intracytoplasmic or intranuclear inclu-
sions. In this review, basic principles common to the field of
amyloid fibril formation and conformational disease are
underlined. Existing models for the mechanism need to be
tested by experiment. The kinetic and energetic bases of the
process are reviewed. The main controversial issue remains
the coexistence of more than one protein conformation. The
possible role of oligomeric intermediates, and of domain-
swapping is also discussed. Mechanisms for cellular defence
and novel therapies are considered.
Keywords: amyloid fibrils; conformational disease; domain
swapping; kinetics; mechanism of fibrillogenesis.
Protein folding is important for cellular events ranging from
transport, accepting and transmitting signals, regulation at
the gene and RNA levels, cell adhesion, changes in
cytoskeleton, metabolic reactions involving various
enzymes, etc. An active protein conformation is needed

associated protein involved in stabilizing axonal
microtubules. Other functions include a role in signal
transduction, and anchoring various kinases and phospha-
tases [9]. Importantly, an anti-amyloidogenic protein,
gelsolin, has been found in plasma and central system fluid
(CSF). This secretory protein is able, by making complexes
with Ab, to inhibit fibril formation and even to break down
already formed fibrils [10]. Recently, it has been found that
the endopeptidase ÔneprilysinÕ degrades Ab peptide. In
neprilysin gene-disrupted mice Ab was found to accumu-
late, with the highest levels in the hippocampus [11].
In Parkinson’s disease, which is the second most common
neurodegenerative disease, several proteins are implicated,
a-synuclein, synphilin (an a-synuclein inteacting protein)
andparkin[12].a-Synuclein is a small (140 amino acid)
acidic protein. It is a naturally unfolded, intracellular and
presynaptic polypeptide that becomes partly helical on
binding to synaptic vesicles [13]. Its function may be, among
others, regulation of synaptic vesicles and neurotransmitter
release [13]. It is interesting that a-synuclein is a target of
serine/threonine [14] as well as tyrosine [15,16] kinases. A
hallmark of Parkinson’s disease is the presence of Lewy
bodies, which are found in sporadic cases of Parkinson’s
disease, in dementia with Lewy bodies and in the Lewy body
variant of Alzheimer’s disease [17]. a-Synuclein is the main
component of the Lewy bodies [18]. Both a-synuclein and
synphilin are required for formation of the Lewy bodies
where ubiquitination of synphilin probably takes place
[12,17]. Parkin is a 465-amino-acid ubiquitin-protein ligase
[17,19]. Mutations in parkin and a-synuclein, in familial

The term ÔamyloidÕ was introduced in 1854 by the
German physician R. Virchow, who named it in the belief
that the iodine-staining component was starch-like [22,23].
The first criterion for detecting amyloid ex vivo was
birefringence of the histological dye Congo Red, observed
under polarized light. As the second criterion, electron
microscopy showed that all amyloid deposits exhibited a
similar fibrillar, submicroscopic structure, bundles of
straight, rigid fibrils ranging in width from 60 to 130 A
˚
and in length from 1000 to 16000 A
˚
[23]. In addition to the
fibrillar component of amyloid, nonfibrillar components
were always found, including serum amyloid protein,
heparan sulfate proteoglycans and apolipoprotein E [23].
The importance of the nonprotein and nonfibrillar compo-
nents of amyloid as observed in vivo remains to be
determined. In vitro studies of the disease related proteins,
as well as other amyloidogenic proteins, have been
concerned mostly with the morphology and kinetics of
fibrillogenesis.
It was concluded by Soto [20] that the pathogenesis of all
the conformational diseases, including prion disease,
involves conformational changes leading to aberrantly
folded proteins, rich in b secondary structure that have a
high tendency to form aggregates and are quite resistant to
proteolysis [20,24]. The field is characterized by several
scientific findings that challenge some of the commonly held
dogmas in biology [24]. These findings are that a protein can

proteins which are initially unfolded or predominantly
b sheet [40–42] and which fold through an a helical
intermediate [43–46].
In vitro, variation of solvent conditions by changing pH
or adding organic solvents [47] can lead to partial unfolding
Table 1. Protein fibrillar inclusions in neurodegenerative and other types of diseases. Data from [2,100]. TSE, transmissible spongiform
encephalopathies.
Disease Protein component Cellular inclusion
Neurodegenerative
Alzheimer’s sau, A42b peptide Neurofibrillary tangles
Pick’s sau Pick bodies/cytoplasmic
Progressive supranuclear palsy (PSP) sau, heat shock proteins Neurofibrillary tangles
Dementia with Lewy bodies a-Synuclein Lewy bodies/cytoplasmic
Parkinson’s a-Synuclein, crystallins Neurofilaments/cytoplasmic
Huntington’s Expanded Glu repeats of Intranuclear inclusion
huntingtin
Spinocerebellar ataxias (SCA) Expanded Glu repeats of Intranuclear inclusion
ataxins 1,3,7
TSE Prion protein, cathepsin B Endosome-like organelles
System amyloidosis
Diabetes type 2 Amylin
Haemodialysis related A b-2 Microglobulin
Reactive amyloidosis Amyloid A
Cystic fibrosis CFTR protein
Ó FEBS 2002 Amyloid-fibrils and conformational disease (Eur. J. Biochem. 269) 3363
and subsequent protein fibril formation [29,48]. With
unfolded polypeptides, partial folding can be obtained by
lowering pH or by heating [39]. In vivo, partial unfolding
may happen as a consequence of lowered protein stability
due to mutation, local change in pH at membranes,

fibrils are [58], b strands (separated by 4.7 A
˚
) running
perpendicular to the long axis of the fibrils and b sheets
extending parallel to this axis. The bstrands form a
b helical twist with the usual repeat at every 115 or 250 A
˚
[56,58]. There are two main types of the fibrils, type 2
fibrils are built from two intertwined filaments, with a
diameter from 80 to 130 A
˚
. Type 1 fibrils are thinner and
are formed from one filament only. There are other types
of fibrils [62]; for example, a fibril and untwisted filaments
of human stefin B [31] (type I cystatin) are illustrated in
Fig. 1.
ENERGETIC AND KINETIC BASIS
OF FIBRILLOGENESIS
The molecular and energetic basis of protein misfolding and
amyloid fibrillogenesis is still largely unknown [20,63]. In the
conclusion to their review, Rochet & Lansbury [35] propose
that future research should be directed towards understand-
ing the mechanism of amyloid-fibril formation, including
environmental factors, such as temperature, ionic strength,
pH and oxidation potential. Proteins have been treated as
an ensemble of rapidly interconverting conformational
substates. In contrast, recent studies have shown that
interconversion between different conformations may be
slow (taking hours to days). For certain proteins the folding
appears to be determined by kinetic rather than thermody-

ÔfilamentsÕ. Fully grown fibrils are then made from one or
more filaments added laterally or, end by end [69]. The
events in the lag-phase are especially important and some
results have been obtained by real time AFM [70,71].
Presence of prefibrillar (oligomeric) intermediates is an
emerging theme [68,72].
The kinetics of fibrillogenesis have been studied by light
scattering [67,72]. Teplow and coauthors [67] have detec-
ted the following steps: (a) peptide micelles form above a
certain critical concentration, (b) fibrils nucleate within
these micelles or on heterogenous nuclei (seeds), and (c)
fibrils grow by irreversible binding of monomers to the
fibril ends. Simpler, colorimetric methods exist for detect-
ing amyloid fibrils. Use of histological dyes Congo Red
[73] and Thioflavin T [74] is widespread. In fact, both dyes
may actually label the filaments better than the fibrils
(E. Z
ˇ
erovnik, unpublished observation). Thioflavin T
fluorescence is a suitable method to follow the kinetics
of fibril formation in an interrupted manner, whereas
interference with the process on longer standing would be
expected. Whether Congo Red is fibril specific has been
questioned [75]. Substances based on Congo Red dye
structure have been used to inhibit fibril formation in vivo
[76] and others based on Thioflavin dye structure to label
the amyloid plaques in brain imaging [77].
Teplow and coworkers [40] have recently reported that an
intermediate with additional a helix structure was shown to
be a key step in Ab fibrillogenesis. The a helical content (as

[69], the kinetics of fibril-growth of two rationally designed
peptides have been compared. One peptide was made more
hydrophobic by replacing Glu by Phe and Trp residues. At
100 l
M
concentration this peptide formed b sheet ribbons
and at a concentration of > 600 l
M
the ribbons were
transformed into rigid fibrils. Due to the balance of weak
forces, fibril and fibre formation is characterized by slow
kinetics. In the particular case [69], fibril formation takes up
to several weeks to complete, as monitored by CD and
TEM.
Serio et al. [79] have studied the yeast prion, sup 35.
Detailed kinetics showed that seeding accelerated the fibril
growth while, with no seeds present, a lag phase was
observed. During this phase, smaller fibrils (seeds) form that
allow rapid assembly. The lag time should decrease
exponentially with increasing soluble protein concentration
if the nucleated polymerization model were applicable,
which was not the case. They have therefore proposed a new
model, termed the nucleated conformational conversion
(NCC) model, which states that oligomers lacking a
conformation leading to fibril formation accumulate and
associate with the nuclei where conformational conversion
takes place as a rate-determining step.
Several other mechanistic models, in addition to the NCC
model, have been proposed: the monomer-derived conver-
sion (MDC) model [60], which is similar to the template

lateral and end-to-end association of the filaments [80]. We
believe that it would be possible to include irreversible
domain-swapped dimers (A-state dimers) in the model,
inplace of monomeric I.
ROLE OF DOMAIN SWAPPING
IN FIBRILLOGENESIS
It is to be noted that several amyloidogenic proteins form
domain swapped-dimers. Such is the case with prion protein
[53], human cystatin C [36,52] and human stefin A [54,82], a
type 1 cystatin. It remains to be seen if these irreversible
transitions, due to high energetic barriers [81,82], have
relevance to amyloidogenesis.
Eisenberg and coworkers have proposed a method by
whichdomain-swappeddimerscouldleadtohigher
oligomerization and amyloid fibrillization [30,81]. If the
exchange of secondary structure elements is not recipro-
cated but propagated along multiple polypeptide chains,
higher order assemblies may form. In principle, any protein
is capable of oligomerization by 3D domain-swapping [83].
By designing an a helical structure that could domain swap,
Eisenberg et al. [84] have shown that it was possible to
design a sequence that permits a reciprocated swap and
another that promotes a propagated swap. Indeed, domain-
swapped dimer and fibrils resulted, as expected. An
interesting observation was also made with ribonuclease
where pair of domain-swapped structures involving N- and
C-terminal parts can coexist. This suggests another possible
mechanism for propagated domain swapping [30].
Staniforth et al. [54] discuss ways in which the domain-
swapped dimer of cystatin could propagate into a fibrillar

It has been suggested by Bergdoll et al.[86]and
confirmed by Itzhaki and coauthors [87] that a proline in
the linker region might facilitate domain swap. It could
rigidify the hinge region and keep it extended [83]. Parallel
reactions in folding have largely been attributed to the
difference in peptide bond configuration at some critical
proline [88] in the denatured state ensemble. This option,
too, should be considered in searching for an explanation
for slow formation of domain-swapped dimers and fibrils.
The energy of activation determined for the lag and growth
phases in a-synuclein fibrillization [39] was  20 kcalÆmol
)1
,
which would be consistent with a proline isomerization
reaction. Of course, there may be other slow events with
high activation energy. It has been found that a slow rate of
unfolding (a high E barrier) prevents amyloid fibril forma-
tion [89] and that fast unfolding leads to increased rate of
fibrillization.
CONNECTION OF PROTEIN FIBRIL
FORMATION TO PATHOPHYSIOLOGY
AND DISEASE
So far, about 20 human proteins have been found in
proteinaceous deposits in various conformational diseases.
These do not demonstrate any sequence or structural
homology. The common event is thought to be a
conformational change, leading to lack of biological
function or gain of toxic activity, and possibly, formation
of amyloid fibrils.
It is a matter of debate as to whether the fibrillar

toxic, causing oxidative stress and, eventually, neural death
[72,93]. The smaller oligomers can interfere with signal
transduction, possibly binding a tyrosine kinase important
for memory formation (long-term synaptic potentiation)
and sau phosphorylation [6].
3366 E. Z
ˇ
erovnik (Eur. J. Biochem. 269) Ó FEBS 2002
In prion diseases [20,24,94], no abundant amyloid
deposition was found in the brain, even though PrP
Sc
(the disease-related conformer of the protein) has a strong
tendency to aggregate in vitro. An interesting observation
was made that PrP
C
(the normal, cellular protein) binds
to survival factors and that the PrP
C
to PrP
Sc
transition
might result in apoptotic cell death. In Huntington’s
disease, activation of microglia following disruption of
neuronal architecture may be the death trigger rather
than the apoptotic pathway [91]. This is consistent with
findings in a transgenic mouse model of Huntington’s
disease, where cell death was neither apoptotic nor
necrotic [92].
MEANS OF NATURAL DEFENCE AND
REGULATION

family of proteins operate as molecules that recruit
chaperones to target proteins. Such diverse proteins as
Bcl2, Raf1, various receptor, transcription factor mole-
cules and Hsp70 compete for binding to members of the
BAG-family of proteins [102]. This binding induces
changes in protein conformation that may have a
profound effect on protein function. Unfortunately,
studying the conformational changes in proteins in vivo
remainsratherelusive.
NOVEL THERAPEUTIC APPROACHES
Novel therapeutic approaches are being directed towards
achieving one of the following goals: either to inhibit and/or
reverse the conformational change, or to dissolve the
smaller aggregates and disassemble the amyloid fibrils.
Several successful attempts have been cited in the literature
including the use of monoclonal antibodies that bind to the
active conformation of the protein and thus inhibit
conformational changes. In Alzheimer’s disease, vacci-
nation is on the horizon, in this case targeting the smaller
oligomers and prefibrillar aggregates [103]. Soto and
coworkers have designed the so called Ômini-chaperonesÕ,
also termed Ôb sheet breakersÕ [20,24], which are peptides
that bind to the sequence of the protein region responsible
for self association. In the prion disease, similarly to
Alzheimer’s, trials are underway using monoclonal anti-
bodies that prevent conformational change [104]. Some
drugs already in use for other purposes have been screened
and several were found that both retard or reverse neuro-
degeneration if used for early intervention and also improve
the disease state in quite desperate cases, as reported by the

(JSI, Ljubljana, Slovenia) is indebted for reading the manuscript, giving
useful comments and editing English. I also thank T. Zavas
ˇ
nik-Bergant
(JSI, Ljubljana, Slovenia) and K. Goldie (EMBL, Heidelberg,
Germany) for taking the TEM picture reproduced in Fig. 1. I am
thankful to M. Ravnikar and M. Pompe-Novak (both National
Institute of Biology, Ljubljana) and I. Mus
ˇ
evic and M. S
ˇ
karabot
(Department of Physics, JSI, Ljubljana) for continuous TEM and
AFM work on human stefins. My gratitude goes to Professor V. Turk
and his team: L. Kroon-Z
ˇ
itko and M. Kenig (at JSI, Ljubljana), for
preparing the recombinant stefins. The author additionally thanks J. P.
Waltho for the model of cystatin A–stefin A dimer reproduced in
Fig. 2B, and to R. A Staniforth (Krebs Institute, University of
Sheffield, UK) for reading the manuscript and giving useful sugges-
tions.
Ó FEBS 2002 Amyloid-fibrils and conformational disease (Eur. J. Biochem. 269) 3367
REFERENCES
1.Raso,S.W.&King,J.K.(2000)Proteinfoldingandhuman
disease. In Mechanisms of Protein Folding (Pain R.H., ed.)
Frontiers in Molecular Biology Series, pp. 406–428. Oxford
University Press, Oxford.
2. Goedert, M., Spillantini, M.G. & Davies, S.W. (1998) Fila-
mentous nerve cell inclusions in neurodegenerative diseases. Curr.

13. Murray, I.V.J., Lee, V.M Y. & Trojanowski, J.Q. (2001) Synu-
cleinopathies: a pathological and molecular review. Clin.
Neurosci. Res. 1, 455–455.
14. Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M.,
Iwatsubo, T., Meijer, L., Kahle, P.J. & Haass, C. (2000) Con-
stitutive phosphorylation of the Parkinson’s disease associated
a-synuclein. J. Biol. Chem. 275, 390–397.
15. Ellis, C.E., Schwartzberg, P.L., Grider, T.L., Fink, D.W. &
Nussbaum, R.L. (2001) a-Synuclein is phosphorylated by mem-
bers of the Src family of protein-tyrosine kinases. J. Biol. Chem.
276, 3879–3884.
16. Nakamura, T., Yamashita, H., Takahashi, T. & Nakamura, S.
(2001) Activated Fyn phosphorylates a-synuclein at tyrosine
residue 125. Biochem. Biophys. Res. Commun. 280, 1085–1092.
17. Chung, K.K., Zhang, Y., Lim, K.L., Tanaka, Y., Huang, H.,
Gao, J., Ross, C.A., Dawson, V.L. & Dawson, T.M. (2001)
Parkin ubiquitinates the a-synuclein-interacting protein, synphi-
lin-1: implications for Lewy-body formation in Parkinson
disease. Nat. Med. 7, 1144–1150.
18. Spillantini, M.G., Schmidt, M.L., Lee, V.M., Trojanowski, J.Q.,
Jakes, R. & Goedert, M. (1997) a-Synuclein in Lewy bodies.
Nature 388, 839–840.
19. Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S.,
Minoshima, S., Shimizu, N., Iwai, K., Chiba, T., Tanaka, K. &
Suzuki, T. (2000) Familial Parkinson disease gene product,
parkin, is a ubiquitin-protein ligase. Nat. Genet. 25, 302–305.
20. Soto, C. (2001) Protein misfolding and disease; protein refolding
and therapy. FEBS Lett. 498, 204–207.
21. Cohen, F.E. (2000) Prions, peptides and protein misfolding. Mol.
Med. Today 6, 292–293.

aggregates of a plant protein: a case of a sweet-tasting protein,
monellin. FEBS Lett. 454, 122–126.
29. Chiti, F., Webster, P., Taddei, N., Clark, A., Stefani, M., Ram-
poni, G. & Dobson, C.M. (1999) Designing conditions for in
vitro formation of amyloid protofilaments and fibrils. Proc. Natl
Acad.Sci.USA96, 3590–3594.
30. Liu, Y., Gotte, G., Libonati, M. & Eisenberg, D. (2001) A
domain-swapped RNase A dimer with implications for amyloid
formation. Nat. Struct. Biol. 8, 211–214.
31. Z
ˇ
erovnik, E., Pompe-Novak, M., S
ˇ
karabot, M., Ravnikar, M.,
Mus
ˇ
evic,I.&Turk,V.(2002)HumanstefinBreadilyforms
amyloid fibrils in vitro. Biochim. Biophys. Acta 1594,1–5.
32. Fa
¨
ndrich, M., Fletcher, M.A. & Dobson, C.M. (2001) Amyloid
fibrils from muscle myoglobin. Nature 410, 165–166.
33. Pertinhez, T.A., Bouchard, M., Tomlinson, E.J., Wain, R.,
Ferguson, S.J., Dobson, C.M. & Smith, L.J. (2001) Amyloid fibril
formation by a helical cytochrome. FEBS Lett. 495, 184–186.
34. Dobson, C.M. (1999) Protein misfolding, evolution and disease.
Trends Biochem. Sci. 24, 329–332.
35. Rochet, J.C. & Lansbury, P.T. Jr (2000) Amyloid fibrillogenesis:
themes and variations. Curr. Opin. Struct. Biol. 10, 60–68.
36. Ekiel, I. & Abrahamson, M. (1996) Folding-related dimerization

46. Z
ˇ
erovnik, E., Virden, R., Jerala, R., Kroon-Z
ˇ
itko, L., Turk, V. &
Waltho, J.P. (1999) Differences in the effects of TFE on the
folding pathways of human stefins A and B. Proteins 36, 205–216.
47. Buck, M. (1998) Trifluoroethanol and colleagues: cosolvents
come of age. Recent studies with peptides and proteins. Q. Rev.
Biophys. 31, 297–355.
48. Chiti, F., Taddei, N., Webster, P., Hamada, D., Fiaschi, T.,
Ramponi, G. & Dobson, C.M. (1999) Acceleration of the folding
of acylphosphatase by stabilisation of local secondary structure.
Nat. Struct. Biol. 6, 380–387.
49. Liu, K., Cho, H.S., Lashuel, H.A., Kelly, J.W. & Wemmer, D.E.
(2000) A glimpse of a possible amyloidogenic intermediate of
transthyretin. Nat. Struct. Biol. 7, 754–757.
50. Hosszu, L.L., Baxter, N.J., Jackson, G.S., Power, A., Clarke,
A.R., Waltho, J.P., Craven, C.J. & Collinge, J. (1999) Structural
mobility of the human prion protein probed by backbone
hydrogen exchange. Nat. Struct. Biol. 6, 740–743.
51. Nettleton, E.J., Tito, P., Sunde, M., Bouchard, M., Dobson,
C.M. & Robinson, C.V. (2000) Characterization of the oligo-
meric states of insulin in self-assembly and amyloid fibril
formation by mass spectrometry. Biophys. J. 79, 1053–1065.
52. Janowski, R., Kozak, M., Jankowska, E., Grzonka, Z., Grubb,
A., Abrahamson, M. & Jaskolski, M. (2001) Human cystatin C,
an amyloidogenic protein, dimerizes through three-dimensional
domain swapping. Nat. Struct. Biol. 8, 316–320.
53. Knaus, K.J., Morillas, M., Swietnicki, W., Malone, M., Surewicz,

cursor to amyloidosis: a discussion of the occurrence, role, and
implications. In Molecular Chaperones in the Cell (Lund, P., ed.),
Frontiers in Molecular Biology Series, pp. 257–278. Oxford
University Press, Oxford.
63. Carrel, R.W. & Gooptu, B. (1998) Conformational changes and
disease – serpins, prions and Alzheimer’s. Curr. Opin. Struct. Biol.
8, 799–809.
64. Ferreira, S.T. & De Felice, F.G. (2001) PABMB Lecture. Protein
dynamics, folding and misfolding: from basic physical chemistry
to human conformational diseases. FEBS Lett. 498, 129–134.
65. Damaschun, G., Damaschun, H., Gast, K. & Zirwer, D. (1999)
Proteins can adopt totally different folded conformations. J. Mol.
Biol. 291, 715–725.
66. Kelly, J.W. (1998) The alternative conformations of amyloido-
genic proteins and their multi-step assembly pathways. Curr.
Opin. Struct. Biol. 8, 101–106.
Ó FEBS 2002 Amyloid-fibrils and conformational disease (Eur. J. Biochem. 269) 3369
67. Lomakin, A., Chung, D.S., Benedek, G.B., Kirschner, D.A. &
Teplow, D.B. (1996) On the nucleation and growth of amyloid
b-protein fibrils. detection of nuclei and quantitation of rate
constants. Proc. Natl Acad. Sci. USA 93, 1125–1129.
68. Harper, J.D., Wong, S.S., Lieber, C.M. & Lansbury, P.T. Jr
(1999) Assembly of A b amyloid protofibrils: an in vitro model for
a possible early event in Alzheimer’s disease. Biochemistry 38,
8972–8980.
69. Aggeli, A., Nyrkova, I.A., Bell, M., Harding, R., Carrick, L.,
McLeish, T.C., Semenov, A.N. & Boden, N. (2001) Hierarchical
self-assembly of chiral rod-like molecules as a model for peptide
b-sheet tapes, ribbons, fibrils, and fibers. Proc. Natl Acad. Sci.
USA 98, 11857–11862.

79. Serio, T.R. & Lindquist, S.L. (2000) Nucleated conformational
conversion and the replication of conformational information by
a prion determinant. Science 289, 1317–1321.
80. Pallitto, M.M. & Murphy, R.M. (2001) A mathematical model of
the kinetics of b-amyloid fibril growth from the denatured state.
Biophys. J. 81, 1805–1822.
81. Schlunegger, M.P., Bennett, M.J. & Eisenberg, D. (1997) Oligo-
mer formation by 3D domain swapping: a model for protein
assembly and misassembly. Adv. Protein Chem. 50, 61–122.
82. Jerala, R. & Z
ˇ
erovnik, E. (1999) Accessing the global minimum
conformation of stefin A dimer by annealing under partially
denaturing conditions. J. Mol. Biol. 291, 1079–1089.
83. Newcomer, M.E. (2002) Protein folding and three-dimensional
domain swapping: a strained relationship? Curr. Opin. Struct.
Biol. 12, 48–53.
84. Ogihara, N.L, Ghirlanda, G., Bryson, J.W., Gingery, M., DeG-
rado, W.F. & Eisenberg, D. (2001) Design of three-dimensional
domain-swapped dimers and fibrous oligomers. Proc. Natl Acad.
Sci. USA 98, 1404–1409.
85. Konno, T. (2001) Multistep nucleus formation and a separate
subunit contribution of the amyloidgenesis of heat-denatured
monellin. Protein Sci. 10, 2093–2101.
86. Bergdoll, M., Remy, M.H., Cagnon, C., Masson, J.M. & Dumas,
P. (1997) Proline-dependent oligomerization with arm exchange.
Structure 5, 391–401.
87. Rousseau, F., Schymkowitz, J.W., Wilkinson, H.R. & Itzhaki,
L.S. (2001) Three-dimensional domain swapping in p13suc1 oc-
curs in the unfolded state and is controlled by conserved proline

generative diseases. Neuron 29, 15–32.
96. Badcoe, I.G., Smith, C.J., Wood, S., Halsall, D.J., Holbrook, J.J.,
Lund, P. & Clarke, A.R. (1991) Binding of a chaperonin to the
folding intermediates of lactate dehydrogenase. Biochemistry 30,
9195–9200.
97. Leroux, M.R. & Hartl, F.U. (2000) Cellular functions of mole-
cular chaperones. In Mechanisms of Protein Folding (Pain R.H.,
ed.) Frontiers in Molecular Biology Series, pp. 364–405. Oxford
University Press, Oxford.
98. Mogk, A., Bukau, B. & Deuerling, E. (2001) Cellular functions of
cytosolic E.coli chaperones. In Molecular Chaperones in the Cell
(Lund, P., ed.) Frontiers in Molecular Biology Series, pp. 1–34.
Oxford University Press, Oxford.
99. Glover, J.R. & Lindquist, S. (1998) Hsp104, Hsp70, and Hsp40: a
novel chaperone system that rescues previously aggregated
proteins. Cell 94, 73–82.
100. Alves-Rodrigues, A., Gregori, L. & Figueiredo-Pereira, M.E.
(1998) Ubiquitin, cellular inclusions and their role in neurode-
generation. Trends Neurosci. 21, 516–552.
101. Garrido, C., Gurbuxani, S., Ravagnan, L. & Kroemer, G. (2001)
Heat shock proteins: endogenous modulators of apoptotic cell
death. Biochem. Biophys. Res. Commun. 286, 433–442.
102. Takayama, S. & Reed, J.C. (2001) Molecular chaperone targeting
and regulation by BAG family proteins. Nat. Cell. Biol. 3, E237–
E241.
103. Ingram, D.K. (2001) Vaccine development for Alzheimer’s
disease:ashotofgoodnews.Trends Neurosci. 24, 305–307.
104. Jones, R. (2001) Blocking prion conversion. Highlights. Nat. Rev.
Neurosci. 2, 605.
3370 E. Z