review

Amyloid formation by globular proteins

under native conditions
Fabrizio Chiti1 & Christopher M Dobson2

The conversion of proteins from their soluble states into well-organized fibrillar aggregates is associated with a wide range
of pathological conditions, including neurodegenerative diseases and systemic amyloidoses. In this review, we discuss the
mechanism of aggregation of globular proteins under conditions in which they are initially folded. Although a conformational
change of the native state is generally necessary to initiate aggregation, we show that a transition across the major energy barrier
for unfolding is not essential and that aggregation may well be initiated from locally unfolded states that become accessible, for
© 2009 Nature America, Inc. All rights reserved.

example, via thermal fluctuations occurring under physiological conditions. We review recent evidence on this topic and discuss
its significance for understanding the onset and potential inhibition of protein aggregation in the context of diseases.

A wide range of human pathologies are associated with the conversion
of polypeptide chains from their soluble states into well-organized fibril-
lar aggregates having extensive β-sheet structure1–3. Such disorders,
which may collectively be grouped as “protein deposition diseases,”
include neurodegenerative conditions, such as Alzheimer’s disease,
Parkinson’s disease and the spongiform encephalopathies. They also
include systemic amyloidoses, such as light chain and lysozyme amy-
loidoses, familial amyloid polyneuropathy and dialysis-related amy-
loidosis, as well as localized amyloidoses, including type II diabetes
and aortic medial amyloidosis. Overall, more than 40 human protein
deposition diseases have been described so far, each having a distinct
clinical profile and each associated with the aggregation of a single
dominant peptide or protein3.
Some of the protein deposition disorders result from the aggregation
of unstructured peptides, intrinsically disordered proteins or unfolded
fragments of otherwise folded proteins3. Many of the polypeptide chains
undergoing aggregation in protein deposition diseases are, however,
globular proteins—that is, they normally adopt under physiological con-
ditions a cooperative and persistent fold in which well-defined secondary
and tertiary structures are present. Examples include immunoglobulin
light chains, transthyretin, β2-microglobulin, lysozyme, various forms of
ataxins, superoxide dismutase 1 and prolactin3. In these highly evolved
states, the propensity of the proteins to aggregate is generally very low4.
Nevertheless, such folded proteins, and indeed many similar proteins
that are not associated with disease, have been shown in vitro to undergo
amyloid fibril formation readily under solution conditions that promote
their partial unfolding, such as at low pH5,6, high temperature7,8, high
pressure9,10 and in the presence of co-solvents11,12. In addition, most
of the mutations associated with hereditary forms of protein deposition
1Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale
Morgagni 50, I-50134 Firenze, Italy. 2 Department of Chemistry, University of
Cambridge, Lensfield Road, Cambridge CB2 1EW, UK. Correspondence should
be addressed to F.C. (fabrizio.chiti@unifi.it) or C.M.D. (cmd44@cam.ac.uk).
Published online 16 December 2008; doi:10.1038/nchembio.131
diseases have been shown to decrease the conformational stability of
the globular native fold (∆GU-F) and to promote aggregation in vitro;
they have therefore been considered to give rise to disease as a conse-
quence of their destabilizing effect13–16. These lines of evidence have
contributed to the formulation of the conformational change hypothesis,
which is based on the simple concept that a process of global or partial
unfolding is required to initiate the aggregation of a globular protein
that is normally cooperatively folded17,18.
This concept can be described by using established thermodynamic
and kinetic principles (Fig. 1a), as well as more recent representations of
protein (un)folding processes based on energy landscapes (Fig. 1b). If a
mutation, or a change of solution conditions, destabilizes and increases
the free energy of the native fold (N) relative to that of the fully unfolded
state (U) or of any partially unfolded state (I) that exists before the
major free energy barrier for folding, it will result in a higher equilib-
rium population of the non-native U and I states. In addition, if such a
mutational or environmental change does not destabilize the transition
state, or destabilizes it to a lesser extent than it does the native state, it
will also result in an increased kinetic accessibility of the U and I states.
Because in such states many of the hydrophobic and backbone moieties
that are normally buried in the interior of the native fold are solvent
exposed, and thus accessible for intermolecular interactions, this readier
thermodynamic and kinetic accessibility of the non-native states will
result in a greatly enhanced propensity for aggregation.
The attainment of the U and I states requires a transition from the
N state across the major free energy barrier for unfolding. This model
of protein aggregation has led for nearly two decades to an extensive
investigation of protein aggregation processes when such conforma-
tional states are populated. Such studies have provided important and
fundamental insights into the nature of the aggregates involved and the
mechanisms of their formation. Nevertheless, recent findings suggest
that unfolding processes of this magnitude are not essential to generate
precursor states that are prone to amyloid formation. Conformational
states thermodynamically distinct from the native state, but structur-
ally similar to it, can be accessed directly from the native state through

nature chemical biology volume 5 number 1 januarY 2009 15

 review
b Human lysozyme
a
TS U D67H single mutations or the F57I T70N or W112R T70N double
I
Energy
Free
U relevant conditions of pH and temperature26,27 (Fig. 2). That this transi-
I tion does not involve crossing the major free energy barrier for unfold-
N*
N*
N range by measurements at high GdnHCl concentrations followed by
N
Number
Number of
of native
Protein aggregation residue contacts
interactions
Figure 1 The process of protein folding. (a,b) Folding is shown according
to classical thermodynamic and kinetic principles (a) and a free energy
landscape (b). The unfolded state (U), consisting of a large ensemble of
unstructured conformations, can collapse to a partially folded state (I)
and then fold across the major free energy barrier for folding to the native
folded state (N). Both figures can be read backward with N unfolding to
form I through the major free energy barrier for unfolding. One or more
locally unfolded states (N*) may become accessible from N via thermal
© 2009 Nature America, Inc. All rights reserved.
fluctuations. Such conformational ensembles represent high energy states
with respect to N under physiological conditions, but are typically separated
from N by a low energy barrier. Although they form transiently and are rarely
populated at equilibrium, they are sampled frequently. Protein molecules
adopting the U, I and N* states or conformations can all self-assemble
and consequently trigger amyloid formation. However, under physiological
conditions, N* is both thermodynamically and kinetically more readily
accessible from N than are I and U and may therefore represent a key
precursor to protein aggregation.
thermal fluctuations. These conformational states, which we term N*,
are at higher energy than N, but are separated from it by a relatively low
energy barrier (Fig. 1a,b). They are therefore only transiently populated
under physiological conditions, yet they can be sampled more frequently
than states such as I and U. The existence of such conformational states
is suggested both by simulations19,20 and by the long-standing observa-
tion that some (often many) of the amide hydrogens buried in the interior
of a native protein can often exchange with the solvent at detectable
rates under physiological conditions, more rapidly than unfolding21.
If such states prove to be of significance for aggregation in vivo, they
could provide a general mechanism for pathological amyloid formation
that does not require the existence of the relatively harsh conditions that
have been observed to give rise to aggregation in vitro.
In this review we summarize the evidence for the aggregation of nor-
mally globular proteins as a direct consequence of fluctuations from the
native state or other local unfolding events, that are, however, distinct
from global unfolding in that they do not involve crossing the major
energy barrier between N and U or I. This review focuses on systems that
adopt fully folded structures under physiological conditions and does not
discuss the aggregation mechanisms of systems that are intrinsically dis-
ordered, such as the amyloid β peptide and α-synuclein, or that contain
unstructured domains, such as the prion proteins. We will focus mainly
on five well-studied proteins: lysozyme, a hyperthermophilic acylphos-
phatase, superoxide dismutase 1, transthyretin and β2-microglobulin.
We show that locally unfolded states may not only be populated under
conditions close to physiological, but are also able to give rise to amyloid
formation. We suggest that the accessibility of an amyloidogenic state
can occur through thermal fluctuations of the native folded state and
that this is likely to be an important step in the pathogenesis of protein
deposition diseases associated with initially globular proteins.
16 volume 5 number 1 JanuarY 2009 nature chemical biology
Variants of human lysozyme, bearing either the I56T, F57I, W64R or
mutations, are associated with familial forms of systemic or renal
amyloidosis22–25. It has been shown through detailed in vitro studies
of the I56T and D67H variants of human lysozyme that these species
undergo transient locally cooperative unfolding under physiologically
ing can be deduced from the rate constant determined for the global
unfolding process, which is estimated to be within the 10−6 to 10−9 s–1
extrapolation to physiological conditions (assuming typical values for
the dependence of the unfolding rate constant on GdnHCl concentra-
tion and temperature)28. By contrast, a value of about 10−1 s–1 has been
determined by hydrogen/deuterium (H/D) exchange experiments for the
“opening” rate constant that gives rise to the partially unfolded ensemble
through fluctuations26. This partially unfolded state is thus accessed at
least five orders of magnitude faster than global unfolding, and must
therefore result from inherent fluctuations associated with the native
state. However, this timescale is very long compared to that of ther-
mal fluctuation occurring within native state ensembles. The partially
unfolded state is therefore formed through a locally cooperative process
and can be considered to be a conformational state thermodynamically
distinct from the native state, but still on the native side of the free
energy barrier for unfolding (N* in Fig. 1).
But is this conformational ensemble amyloidogenic? H/D exchange
rate measurements monitored by means of NMR spectroscopy have
shown that in this conformational ensemble the β-domain and C-helix,
spanning approximately residues 36-102, are simultaneously highly
unstructured, whereas the remainder of the α-domain appears to main-
tain its native-like structure26,27 (Fig. 2). The region of the sequence
exposed in this way has been shown to be highly amyloidogenic: pro-
teolysis with pepsin of fibrils from human lysozyme reveals that the
minimal region of the sequence left undigested, and thus presumed to
form the structured core of the fibrils, encompasses residues 32–108
(ref. 29) (Fig. 2). The same structural fluctuations from the native state
to the partially unfolded ensemble also occur for the wild-type protein
and for a non-amyloidogenic variant, bearing the T70N substitution,
Structural
fluctuations
Native monomer (N) Locally unfolded Amyloid fibrils
monomer (N*)
Figure 2 Proposed process of fibril formation for human lysozyme. The
protein is initially in its native monomeric form N (left, Protein Data Bank
(PDB) entry 1LZ1). In this structure, the region spanning residues 36–102,
encompassing the β-domain and the C-helix, is indicated in light blue. A
locally unfolded monomer N*, having a highly flexible β-domain and C-helix,
is accessed through thermal fluctuations26,27. The transition from N to N*
is distinct from the global unfolding process and is facilitated by the D67H
and I56T mutations associated with familial amyloidosis. The region that is
unfolded in N* promotes the process of aggregation and is found to form the
β-sheet core in the amyloid fibrils29 (right). The picture of the amyloid fibril
reported in the figure is for descriptive purposes only and emphasizes simply
that the β-sheet core of the fibrils is contributed by the region unfolded in N*.

that is present in about 10% of the human population30. However, the
kinetic data show that these aggregation-prone states are accessed orders
of magnitude less frequently than those in the amyloidogenic variants,
unless the temperature is raised to values well above physiological30.
These findings allow a mechanism of amyloid fibril formation to be
proposed for the I56T and D67H variants of human lysozyme31. Full
unfolding to form states such as U or I across the major energy barrier
for unfolding is not required32. The variants access an N* state on the
“native side” of the barrier through thermal fluctuations that are rare
in the wild-type protein, but much more frequent in the amyloidogenic
variants31 (Fig. 2). Fibril formation will occur as a consequence of
the intermolecular interactions between regions that are unfolded and
exposed in the N* state; such regions will thus form the cross-β core of
the resulting fibrils (Fig. 2). Because the fluctuations from the native
to the N* state will occur with significant frequency under physiologi-
cal conditions, a mechanism of this type does not need to invoke even
transiently the existence in vivo of extreme conditions (for example,
low pH or high temperature) in the pathogenesis of familial lysozyme
amyloidosis.
Acylphosphatase from Sulfolobus solfataricus
If a globular protein can aggregate via local structural fluctuations that
© 2009 Nature America, Inc. All rights reserved.
are independent of complete unfolding, it might be expected that (in
some cases at least) the initially formed oligomeric species could retain
a native-like structure. The study of acylphosphatase from Sulfolobus
solfataricus (Sso AcP), a protein that has no link with human disease but
that has proved to be a valuable system for the study of the mechanism
of protein aggregation, has been particularly informative in this context.
Aggregation of Sso AcP has been characterized in particular detail in
a buffered solution containing 20% (v/v) trifluoroethanol (TFE)32–34.
Under these conditions, before aggregation, Sso AcP is present in a fully
native-like state, as indicated by far- and near-UV circular dichroism,
enzymatic activity measurements and stopped-flow measurements of
folding and unfolding rates32–34.
Aggregation of initially folded Sso AcP under these conditions
has been found to occur in two distinct and sequential kinetic steps33
(Fig. 3). In the first step, the protein converts within one minute from the
initial native state into small oligomeric aggregates33. These oligomers
(i) have a secondary structure content close to that of the native state, as
revealed by far-UV circular dichroism and Fourier-transform infrared
spectroscopy, (ii) do not yet bind Congo red, thioflavin T or 8-anili-
nonaphthalene-1-sulfonate (ANS), and remarkably (iii) have extensive
enzymatic activity33. In the second step, these native-like aggregates
convert, on a timescale of several minutes, into spherical species and
chain-like protofibrils with diameters of 3 to 5 nm, which bind Congo
red, thioflavin T and ANS and have an extensive β-sheet structure and
no enzymatic activity33.
Under the conditions used to stimulate aggregation in these studies,
the unfolding process of Sso AcP is approximately four and two orders
of magnitude slower, respectively, than the first and second aggrega-
tion steps, which indicates that global unfolding cannot be a neces-
sary prerequisite of the initiation of aggregation32,33. The first step of
aggregation of initially native Sso AcP therefore results in oligomeric
species that maintain a native-like structure (Fig. 3). Protein engineer-
ing studies have established that this assembly process is promoted
by interactions between the unstructured N-terminal segment and a
portion of the globular unit, most probably the peripheral β-strand 4
(refs. 34–36) (Fig. 3).
Although formation of the initial native-like aggregates does not
require global unfolding, it does not occur at a detectable rate except in
the presence of a co-solvent, such as 20% TFE. Kinetic measurements of
nature chemical biology volume 5 number 1 januarY 2009 17
review
N
Structural
fluctuations
N*
Native Native-like Native-like Amyloid-like
monomer (N) monomer (N*) aggregates protofibrils
Figure 3 Proposed process of aggregation for Sso AcP. The protein is
initially in its native monomeric form N (left, PDB entry 1Y9O). In this
state the unstructured N-terminal segment and β-strand 4 are depicted in
blue and red, respectively. A native-like monomer N* is accessed through
thermal fluctuations32–34. The transition from N to N* is distinct from the
global unfolding process across the major free energy barrier and is aided
by mutations destabilizing the native state N. Interactions between the
unstructured N-terminal segment and a portion of the globular structure,
most probably the peripheral β-strand 4, promote the initial formation of
native-like aggregates in which the various protein molecules maintain a
native-like topology34–36. These aggregates then convert into protofibrils with
amyloid-like characteristics (right). Reprinted from ref. 36 with permission
from Elsevier.
H/D exchange using mass spectrometry reveal that Sso AcP undergoes
more rapid fluctuations in the presence of TFE than in simple aqueous
solutions34. It is therefore likely that, in the presence of the co-solvent,
Sso AcP acquires a sufficiently high dynamic character to allow confor-
mational ensembles such as N* to be sampled more frequently than is
the case in its absence (Figs. 1 and 3). Nevertheless, dynamic behavior
of this type will be a characteristic of the protein under physiological
conditions. As a result, the globular unit of Sso AcP is likely to interact
with the unstructured N-terminal segment and give rise to the formation
of initial aggregates that convert later into amyloid-like protofibrils via
internal reorganization (Fig. 3).
Human superoxide dismutase 1
The lysozyme and Sso AcP cases described above have shown that N*
states can be accessed transiently from N structures through fluctua-
tions, and that the frequency of the fluctuations increases with factors
destabilizing the N state, such as mutations or co-solvents. In principle,
however, locally destabilizing mutations may also cause the persistent,
rather than transient, formation of conformations with locally unstruc-
tured regions in the absence of global unfolding (N*). This situation
will occur if the mutations destabilize the N state in such a manner
that its stability is lower than one or more N* states, and the latter then
become the predominant species at equilibrium under physiological
conditions. This appears to be the case for the S134N mutant of copper-
zinc superoxide dismutase 1 (SOD1), a variant associated with familial
amyotrophic lateral sclerosis (FALS)37. S134N SOD1 has been found to
have an essentially native-like dimeric structure, very similar to that of
wild-type SOD1 (refs. 38,39); nevertheless, in S134N SOD1 the region
of the sequence including the mutation site, known as loop VII or the
“electrostatic loop,” has been shown to be highly flexible and structurally
heterogeneous in solution, as revealed by NMR, even in the dimeric and
fully metal-bound form39 (Fig. 4).
Multiple lines of evidence indicate that this unstructured loop plays
a key role in aggregation of S134N SOD1 (refs. 38,39). First, NMR
studies have shown that the dimeric and fully metal-bound S134N
SOD1 mutant can form transient, soluble oligomers through interac-
tions involving the unstructured loop while the remainder of the mol-
ecule maintains its native structure39 (Fig. 4). In addition, crystals of the
same variant have been shown to consist of amyloid-like filaments in
 review
which adjacent dimers interact via hydrogen bonding and hydrophobic
contacts contributed by the unstructured loop and the cleft between the
open-ended edge β-strands 5 and 6 (ref. 38) (Fig. 4). In the interface
between adjacent dimers, the loop of a given dimer interacts with the
cleft of the second dimer. The loop of the latter also forms an interaction
with the cleft of the preceding dimer, so that the same type of interac-
tion is present twice (Fig. 4). In such an amyloid-like array, the dimers
have a native-like structure, and the individual β-strands are oriented
approximately perpendicularly to the filament axis38. These results indi-
cate that S134N SOD1 dimers can form oligomers and even fibrils while
maintaining a largely native-like conformation and without the need
to dissociate and unfold before self-assembly. X-ray structures with
disordered loops have been reported for other SOD1 variants, including
H46R SOD1 (ref. 40) and G85R SOD1 (ref. 41).
Mutations associated with FALS have been proposed to exert their
pro-aggregating potential through a variety of mechanisms, for example
by decreasing the conformational stability of the native fold, by increas-
ing the intrinsic aggregation propensity of the sequence, by causing
metal loss, post-translational modifications, dimer dissociation and so
on (ref. 42 and references therein). It is however possible that for some
particular SOD1 variants, such as S134N SOD1, H46R SOD1 and G85R
SOD1, amyloid formation occurs directly from dimeric native-like
© 2009 Nature America, Inc. All rights reserved.
states, which may be populated under physiological conditions.
In another study the folding mechanism of a quadruple mutant of
SOD1 has been investigated, in its monomeric form, in the absence of
any bound metal ions and free cysteine residues (apoSOD1)43. It has been
shown from mutational studies, by means of the Φ value analysis, that
in the folding of apoSOD1 the β-strands 5, 6 and 8 are not yet formed in
the major transition state but form later in the process. This result has led
to the suggestion that an intermediate exists that is close to N along the
reaction coordinate but has a high free energy relative to the native pro-
tein43. This “late intermediate” state appears to be characterized by some
unfolded regions, but also by structured regions that become exposed as
a result of the unfolding of the other regions. Such a conformational state
is accessible through thermal fluctuations of the monomeric apoenzyme
and may be the species that initiates the aggregation reaction43.
Native dimer Oligomer Fibril
Figure 4 Proposed process of fibril formation for S134N SOD1. The
protein is initially in its native dimeric form (left, PDB entry 1OZU), in
which the highly flexible electrostatic loop (blue) and the peripheral open-
ended β-strands 5 and 6 (red) play a key role in aggregation. Native dimers
interact to form larger oligomers stabilized by transient interactions between
electrostatic loops from different dimers39. These oligomers grow further
to form fibrils (right). In the fibrillar structure indicated in the figure,
blue, orange and green colors refer to different native-like dimers38. The
interactions between adjacent dimers occur via hydrogen bonding and
hydrophobic contacts established between the electrostatic loop of a dimer
and a cleft between β-strands 5 and 6 of the next dimer; the electrostatic
loop of the latter forms a similar interaction with the cleft between β-strands
5 and 6 of the preceding dimer38. The fibril structure on the right is
reprinted with permission from ref. 38.
18 volume 5 number 1 JanuarY 2009 nature chemical biology
Human transthyretin
Conversion of both wild-type and mutant transthyretins (TTRs) from
their native tetrameric states into amyloid fibrils is associated with a
range of diseases, including senile systemic amyloidosis (SSA), familial
amyloidotic polyneuropathy (FAP), familial amyloid cardiomyopathy
(FAC), leptomeningeal amyloidosis (LMA), carpal tunnel syndrome
(CTS) and vitreous opacity44. Under nonpathological conditions, wild-
type TTR is normally a homotetramer in which each subunit consists of
eight β-strands (A-H) arranged in a sandwich of two β-sheets, involving
strands DAGH and CBEF, respectively45 (Fig. 5).
It is widely accepted that the native TTR tetramer needs to dis-
sociate into a locally unfolded monomeric state in order to become
competent for amyloid fibril formation46–48 and that this process is
both energetically and kinetically favored by the mutations that are
linked to TTR-associated familial diseases16. However, this conver-
sion to the amyloid-competent form of the protein does not involve full
unfolding of the monomer once it is released from the other subunits
(Fig. 5); indeed, spectroscopic data and limited proteolysis studies indi-
cate that monomeric wild-type TTR maintains largely folded native-like
β-strands AGH and BEF, with only the peripheral β-strands C and D
undergoing substantial unfolding47 (Fig. 5). Thus, H/D exchange mea-
surements, carried out on the TTR tetramer and monitored by using
NMR, indicate that the backbone amide hydrogen atoms of the C and
D strands, and of the loops preceding and following the D strands, are
the least protected from H/D exchange relative to the remaining strands,
which lends further support to the structural lability of this region49,50.
Following crystallization of the A108Y L110E mutant TTR, two crystal
structures differing in the conformation of the D strands were observed,
thereby confirming the ability of the D strand to adopt multiple con-
formations51. As this partially unfolded state maintains a native-like
structure and features only local unfolding, it is distinct from states such
as I and U and bears a close structural resemblance to conformations or
conformational ensembles such as N*.
The structure of the amyloid fibrils formed by TTR has been inves-
tigated in molecular detail in two independent studies using (i) fibrils
generated from spin-labeled protein at pH 4.4 and physiological
temperature52,53 and (ii) fibrils formed from a mutational form of TTR
containing the Y114C substitution, at 55 °C and physiological pH54.
The first study was performed using site-directed spin labeling elec-
tron paramagnetic resonance (SDSL-EPR) to monitor subunit interfaces
in the fibril52,53, and the second was performed using H/D exchange
rate measurements to probe the regions of the sequence involved in
the formation of the β-sheet fibril core54. A structural model of a TTR
fibril resulting from combining the results of these two approaches
consists of an array of consecutive monomers arranged along the axis
of the fibril, in which the C and D strands and the loop connecting
them appear to be unfolded (Fig. 5). The core structure involves the
AGH and BEF sheets of each monomer in a native-like conformation
(Fig. 5). The canonical cross-β structure in the fibril, typical of the
amyloid architecture, results from intermolecular interactions between
B strands and A strands from adjacent molecules (that is, head-to-head
monomer interaction) and between F strands and H strands, also from
adjacent monomers (Fig. 5).
These results suggest that, following tetramer dissociation, the TTR
monomer undergoes only a local unfolding transition, involving princi-
pally the C and D strands, which are both peripheral to their respective
β-sheets47 (Fig. 5). Unlike the lysozyme and SOD1 cases described
above, the interaction between TTR monomers in the fibrils is not medi-
ated by the newly unfolded regions, but rather by the A and B strands
that become solvent exposed following dislocation of the C and D
strands, which remain similarly exposed in the fibrils53,54 (Fig. 5).
 review

Unfolded 115 Å
C and D

Fibril

Native Locally unfolded

tetramer monomer N´

C´ D´
C´

D
C
© 2009 Nature America, Inc. All rights reserved.

C˝ D˝
Structure of three
Structure of two subunits C˝
subunits in the fibril
in the tetramer

N˝

Figure 5 Proposed process of fibril formation for human TTR. The protein is initially in its native tetrameric form (left, PDB entry 1F41). Two subunits of the
tetramer are shown enlarged to illustrate the structural arrangement of the DAGH and CBEF sheets in one subunit and of the D′A′G′H′ and C′B′E′F′ sheets in
the other subunit. The region encompassing β-strands C and D is shown in red in both subunits. Aggregation involves dissociation of the tetramer to form a
monomer in which the C and D strands are unfolded47. The transition from the native tetramer to the locally unfolded monomer is enhanced by the mutations
associated with disease16. The region that is unfolded in the monomer is also unfolded in the fibril52–54 (right). A section of the fibril is shown enlarged to
illustrate the unfolded state of the C and D strands and the orientation of the remaining β-strands. The A and B strands of each subunit form a continuous
hydrogen bonded network with the A and B strands of the preceding subunit. The F and H strands of each subunit also form a continuous hydrogen
bonded network with the F and H strands of the next subunit (right). The fibril structure on the right is adapted with permission of the American Society of
Biochemistry & Molecular Biology from ref. 54. Permission conveyed through Copyright Clearance Center, Inc.

Human β2-microglobulin
Amyloid fibril formation by β2-microglobulin (β2m) is a consequence
of medical treatment in which hemodialysis is used to combat kidney
failure and gives rise to a pathological state known as dialysis-related
amyloidosis55. The pioneering observation that a six-residue truncation
at the N terminus of β2m causes both an increase in structural flexibil-
ity within the native state, as detected by limited proteolysis and H/D
exchange monitored by mass spectrometry and NMR spectroscopy, and
a higher tendency to form amyloid fibrils at physiological pH, suggested
nearly a decade ago that amyloid fibril formation by β2m can involve a
native-like conformational state56.
Subsequently, the investigation of the folding process of full-length
β2m has allowed the identification of at least one intermediate that forms
in the dominant folding pathway after the major energy barrier for fold-
ing57–59. This species has been termed I2 (ref. 57) or IT (refs. 58,59) by
the various authors that have described them. Although it remains to be
elucidated whether I2 and IT represent the same conformational species,
or alternatively are structurally and thermodynamically distinct states,
they both accumulate after the major energy barrier for folding. The
rate of conversion of IT into the fully native state N is determined by
the slow trans (intermediate) to cis (native) conversion of the His31-
Pro32 peptide bond58,59. Interestingly, both I2 and IT have been shown
to remain populated, albeit to a small extent and in equilibrium with
the fully native state, after completion of the folding process59,60. A
conformational state structurally related to IT, termed M*, can be formed
under native conditions following addition of Cu2+ ions61,62. I2, IT and
M* will herein be collectively referred to as N* states, to be consistent
with the nomenclature used in this review.
Evidence exists that such β2m N* states can add, under physiologi-
cal conditions, to preformed fibrils that act to seed59,60 or even initiate
aggregation de novo when formed upon Cu2+ addition61,62. In the latter
case it has been found that Cu2+ binds to the native state N, acceler-
ates the conversion to and stabilizes N*, and promotes its oligomeriza-
tion into dimers, tetramers and hexamers61,63–66. Such oligomers have
a native-like structure, as inferred from NMR and near-UV circular
dichroism spectroscopy61.
The structure of IT has been investigated using NMR and circular
dichroism67. These studies have shown a native-like compactness and
secondary structure content for this state, but also detectable structural
perturbations throughout the sequence67. The structure of this species
has also been determined through studies of the P32G mutant using
NMR spectroscopy59. Because peptide bonds involving nonproline
residues are very unstable when they adopt a cis configuration, this
mutant represents a valuable means of stabilizing N* relative to N,
thus enabling its structural investigation. The NMR analysis of the
P32G mutant revealed that the N terminus, the two peripheral A and
D strands and the two loops connecting the B and C and the F and G
strands have small structural differences from the fully native state59.
In agreement with the NMR structural analysis of the P32G variant,
the crystallographic structure of the P32A mutant shows considerable

nature chemical biology volume 5 number 1 januarY 2009 19

 review
a b Pro32
Asp53
His31
Ala32
Phe30
c 180
Trp60
Phe56 Phe56
Phe30 Trp60 Phe30
Phe62
Tyr63
Leu54Leu54
B E D D E B
alterations in the BC loop and in the peripheral D strand relative to the
© 2009 Nature America, Inc. All rights reserved.
wild-type structure (the D strand has lost the β-bulge that is present in
the wild-type structure)62 (Fig. 6). In addition, some re-packing of the
hydrophobic core of the protein was detected in the mutant relative to
the wild-type protein62 (Fig. 6).
After these studies it is clear that β2m becomes prone to form fibrils
following a structural conversion that is distinct from the global unfold-
ing process. The N* states of β2m characterized so far are neither par-
tially unfolded nor fully unfolded states such as I or U. They coexist
with the fully native state and maintain a largely native-like topology.
The IT and M* states of β2m are separated from N by a relatively high
energy barrier, as a result of cis-trans prolyl peptide bond isomerization.
Nevertheless, the resulting N* states are on the “native side” of the free
energy barrier for unfolding and in this respect are analogous to the N*
states of lysozyme, Sso AcP, SOD1 and TTR.
Factors affecting native state conformational stability
The analysis of the systems described in the previous five sections
suggests that aggregation of at least some globular proteins may well
be initiated by fluctuations giving rise to the population of amy-
loidogenic native-like states, without the need to cross the major
free energy barrier for unfolding. This view is not in any manner
incompatible with the notion that most of the mutations giving rise
to protein deposition diseases, and directly involving the globular
protein that undergoes aggregation, decrease the protein’s free energy
change of unfolding (∆GU-F); mutations or indeed environmental
changes that destabilize the native structure generally increase the
populations of all non-native states, including U, I and N* states. It
is therefore not surprising that inverse correlations have been found
between the free energy change of unfolding (∆GU-F), or a correlated
parameter, and the propensity to aggregate for all five proteins dis-
cussed in this article13,15,16,34,68.
Accordingly, species that bind selectively to the native structure of
such proteins, for example ligands and antibodies, have been shown to
substantially reduce the propensity for aggregation69–72. Such binding
inhibits not only global unfolding but also the magnitude of the thermal
fluctuations of the native ensemble. In fact, small molecules that bind
selectively to the native tetramer of TTR (ref. 69), to the dimeric state
of SOD1 (ref. 70) or to the monomeric state of Sso AcP (ref. 71) have
all been shown to have an inhibitory effect on aggregation. Similarly,
20 volume 5 number 1 JanuarY 2009 nature chemical biology
Figure 6 Superposition of β2m crystallographic structures. Native wild-type
(gray) and folded P32A (green) β2m structures are shown62. The structures
correspond to chain B of PDB entries 2CLR (wild-type) and 2F8O (mutant),
respectively. (a) The β-bulge present at position 53 in the wild-type protein
and absent in the mutant is indicated. (b) The detail of the loop between
β-strands B and C. The His31-Pro32 peptide bond is in cis and trans
configuration in the wild-type and mutant proteins, respectively. The cis-
to-trans conversion causes the repositioning of Phe30 (green arrow in b).
(c) The major structural differences involving the hydrophobic side chains
in the two variants are shown. The crystallographic structure of the P32A
P32A
WT
mutant, and the related NMR solution structure of the P32G mutant, are
thought to be close to both the structure of the Cu2+-bound state62 and
the late intermediate state accumulating during folding and coexisting
with the native state at equilibrium under physiological conditions59. The
concentration of this conformational state, which has been shown to be
highly aggregation-prone, increases during hemodialysis as a consequence of
the increase of both β2m and Cu2+ concentrations59,62. In fact, the increase
of protein concentration increases the concentration of any state populated
at equilibrium, and Cu2+ shifts the equilibrium from the native to the native-
like structure. The figure is reprinted with permission from ref. 62. Copyright
2004 American Chemical Society.
camelid heavy chain antibody fragments have been shown to bind to
the amyloidogenic variants of human lysozyme and inhibit aggregation
substantially by suppressing the local unfolding process and enhancing
the cooperative nature of the native state72. The design of species of
this type therefore represents an important strategy to prevent protein
deposition diseases.
Generalization of the concept of native-like aggregation
We have shown that native-like states expressing limited local perturba-
tions can be accessed transiently via structural fluctuations facilitated by
mutations, more stably via mutations that cause the unfolding of nor-
mally structured regions, or via other processes such as subunit disso-
ciation or cis-to-trans prolyl peptide bond isomerization. The resulting
native-like states can be highly amyloidogenic and may be responsible
for aggregation under physiological conditions. In addition to the sys-
tems described here, other normally folded proteins have been shown
to aggregate in vitro from native-like states, including human insulin73,
the protein S6 from Thermus thermophilus74, human ataxin-3 (ref. 75),
the acylphosphatase from Drosophila melanogaster76 and a mutant of
sperm whale apomyoglobin77.
The resulting aggregates may maintain their native-like structure
in the initial oligomers only and later undergo a global structural
reorganization to form the amyloid structure. We have discussed a
clear example of this behavior (Sso AcP), but an even more marked
example is insulin, which converts from completely helical initial
aggregates to fibrils that have an almost completely β-sheet struc-
ture73. In other cases the aggregates may maintain their native-like
structure even in the fibrillar state, as found for S134N SOD1 and
TTR, as the resulting assemblies contain the essential arrangement
of hydrogen bonds that stabilize the fibrillar form of the polypeptide
chain. The latter situation is naturally much more likely to occur for
proteins that contain only or mainly β-sheet structure (for example,
S134N SOD1, TTR, β2m), while other predominantly helical proteins
require major structural reorganization to form the cross-β structure
typical of the fibrils (for example, lysozyme, Sso AcP and insulin).
Moreover, the newly formed interfaces within the native-like oligom-
ers and fibrils may involve the regions that have become unfolded (for
example, lysozyme and S134N SOD1) or that remain structured but
have been exposed by the unfolding of other portions of the protein
(for example, TTR) in the amyloidogenic native-like state.

Future directions
The observations described here reveal the importance, for understand-
ing protein deposition diseases, of investigating the structure and the
dynamics of monomeric precursor species and the mechanisms by which
they self-assemble under conditions as close to physiological as possible.
The use of denaturing conditions in which the native state is no longer
significantly populated, and where highly unfolded states dominate the
kinetics of protein aggregation, is of great interest for investigating the
nature of the amyloid state in general and for inducing aggregation
processes that would otherwise be extremely slow. Nevertheless, to relate
to in vivo behavior it is essential to probe the native state and its dynam-
ics and to detect, directly or indirectly, locally unfolded conformations
accessed through structural fluctuations from the native state under
physiological conditions.
The characterization of physiologically accessible native-like states
can also allow an understanding of why only a limited number of glob-
ular proteins are associated with protein deposition diseases. It may
well be, for example, that aggregation occurs physiologically when
such states are more easily accessed, whereas in cases where extensive
unfolding and reorganization is needed to produce highly organized
fibrillar assemblies, amyloidogenic locally unfolded states are more
rarely accessed and misfolding may result in clearance rather than
© 2009 Nature America, Inc. All rights reserved.
deposition.
Another important objective in this general field of research is to
understand the interactions between these locally unfolded species and
the molecular chaperones and dedicated quality control processes that
exist within cells to protect against aggregation. We know that such
processes mainly target unfolded or misfolded species, preventing their
aggregation and promoting their folding or degradation. Conformations
such as N* may be less easily recognized and targeted by these systems,
and this may be another reason why certain globular proteins may evade
the cellular surveillance, and, for example, give rise to diseases when
the mutations are significantly but not dramatically destabilizing16,31.
The elucidation of these issues will not only considerably enhance our
knowledge of the manner in which proteins have evolved and function
both normally in health and abnormally in disease, but will also increase
our chances of rationally designing therapeutic strategies against protein
deposition diseases associated with normally folded proteins.
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