Báo cáo khoa học: Amyloid structure – one but not the same: the many levels of fibrillar polymorphism potx

MINIREVIEW
Amyloid structure – one but not the same:
the many levels of fibrillar polymorphism
Jesper S. Pedersen
1
, Christian B. Andersen
2,3
and Daniel E. Otzen
4
1 Department of Biochemistry, Molecular Biology, and Cell Biology, Rice Institute for Biomedical Research,
Northwestern University, Evanston, IL, USA
2 Protein Structure and Biophysics, Novo Nordisk A ⁄ S, Ma
˚
løv, Denmark
3 Institute of Biophysics, National Research Council, CNR, Palermo, Italy
4 Department of Molecular Biology, Center for Insoluble Protein Structures, Interdisciplinary Nanoscience Centre,
University of Aarhus, Denmark
Amyloid in disease and functional
fibrillar structures
Amyloid and amyloid-like structures are normally
associated with several debilitating diseases, including
Alzheimer’s, Parkinson’s, Huntington’s and transmissible
spongiform encephalopathies [1]. In these cases, the
formation of amyloid and other aberrant aggregates
represents an alternative type of folding that makes
Keywords
aggregation; amyloid; fibrillar polymorphism;
glucagon; mechanism; protein folding
Correspondence
J. S. Pedersen, Department of
Biochemistry, Molecular Biology, and Cell

Biology, Rice Institute for Biomedical
Research, Northwestern University,
2205 Tech Drive, Hogan 2-100, Evanston,
IL 60208, USA
Tel: +1 847 881 6617
E-mail:
D. Otzen, Department of Molecular Biology,
Center for Insoluble Protein Structures,
Interdisciplinary Nanoscience Centre,
University of Aarhus, Gustav Wieds Vej 10,
DK-8000 Aarhus C, Denmark
Fax: +45 8612 3178
Tel: +45 8942 5046
E-mail:
(Received 16 April 2010, revised 2
September 2010, accepted 17 September
2010)
doi:10.1111/j.1742-4658.2010.07888.x
Many proteins and peptides can form amyloid-like structures both in vivo
and in vitro. Although strikingly similar fibrillar structures can be observed
across a variety of amino acid sequences, the fibrils formed often exhibit a
stunning wealth of polymorphisms at the level of electron or atomic force
microscopy. This appears to violate the Anfinsen principle seen for globu-
lar proteins, where each protein sequence codes for just one well-defined
fold. To a large extent, polymorphism reflects variable packing of a single
protofilament structure in the mature fibrils. However, we and others have
recently demonstrated that polymorphism can also reflect real structural
differences in the molecular packing of the polypeptide chains leading to
several possible protofilament structures and diverse mature fibrillar struc-
tures. Glucagon has been a particularly useful model system for studying

the fibrillogenesis mechanisms that lead to the formation of structural poly-
morphism, thanks to its single tryptophan residue and the availability of
large quantities at pharmaceutical-grade quality. Combinations of struc-
tural investigations and seed extension experiments have revealed the repro-
ducible formation of at least five different self-propagating fibril types from
subtle variations in growth conditions. These reflect the underlying com-
plexity of the peptide conformational landscape and provide a link to
natively disordered proteins, where structure is dictated by context in the
form of different binding partners. Here we review some of the latest
advances in the study of glucagon fibrillar polymorphism and their implica-
tions for mechanisms of fibril formation in general.
Abbreviations
AFM, atomic force microscopy; EM, electron microscopy; ITC, isothermic titration calorimetry; SAXS, small angle X-ray scattering;
ThT, thioflavin T.
FEBS Journal 277 (2010) 4591–4601 ª 2010 The Authors Journal compilation ª 2010 FEBS 4591
the protein become toxic and ⁄ or lose its natural func-
tion [2–4]. In systematic amyloidoses, it appears to be
the massive accumulation of amyloid per se that is
pathological, leading to organ failure and eventual
death [5–7]. Smaller amounts of amyloids accumulated
in sensitive locations, such as the cornea, can also lead
to severe functional impairment [8]. In most neurode-
negerative diseases, however, prefibrillar aggregates are
the toxic species, as detailed by Stefani in the review in
this series [9].
It has been suggested that the ability to form amy-
loid-like structures, given appropriate conditions, is a
general property of the polypeptide chain [10,11]. This
ability not only has pathological consequences, but can
be turned into good use as functional amyloid, serving

as structural anchoring material for a range of pur-
poses ranging from biofilm formation to a matrix to
anchor melanin as well as for the reduction of interfa-
cial tension, coating of spores and many other as yet
unknown functions (see [12] and references herein).
Several different protein and peptide hormones, includ-
ing insulin and glucagon [13,14], have long been
known to readily form fibrils, and it has recently been
proposed that functional amyloid structures could
serve as in vivo intracellular storage of these hormones
in pituitary secretory granules, stabilized by interac-
tions with glucosaminoglycans [15,16]. Upon release
from the cells, these amyloid structures would gradu-
ally dissociate into active monomers in the blood-
stream [17], which would imply that the amyloid
structures formed by these hormones in vivo should
have evolved to be relatively unstable.
Fibrillar polymorphisms reflect
structural ambiguity in amyloid fibrils
The key to the properties of amyloid assemblies lies
in the regularity and repetitiveness of their underlying
molecular architecture of intermolecular b-strands
stacked perpendicular to the fibril axis [18], often
organized in several b-sheets parallel to the axis.
Although atomic resolution structures of the molecu-
lar packing of peptides in amyloid-like structures are
beginning to emerge [19], much of our understanding
of the structure of amyloid-like fibrils comes from
electron microscopy (EM) techniques and atomic
force microscopy (AFM) [20]. Images produced by

these techniques often show stunningly beautiful, per-
fectly ordered fibrils, some with regular twisting, oth-
ers seemingly smooth and still others as parallel
bundles of two or more protofilaments (Fig. 1). Fibril
preparations often contain several different morpho-
logies, and for some of these the turn lengths and
morphology are directly correlated to the number of
protofilaments they contain [21]. Strikingly similar
fibrillar morphologies can arise from proteins and
peptides with very diverse amino acid sequences [22–
24]. In other cases, particular morphologies can be
generated by manipulating the specific conditions for
fibril formation [25–27].
Initially it was assumed that the protein fold found
in amyloid structures was a single energy minimized
structure following Anfinsen’s single-fold principle for
native proteins [28], with the different morphologies
representing different lateral associations of a single,
lowest-energy protofilament structure [22,29]. The
only exception from this rule was thought to be the
prions, which were shown to form several variants
[30,31], so-called self-propagating strains, with different
Fig. 1. Comparison of representative EM morphologies of the five
different glucagon fibril types, type A [27,44,56,63], B
unagitated
[44,53,56], B
agitated
[23,27,71,73], D [27,73] and S [43,61,73], where
combinations of spectroscopic, thermostability and seed extension
kinetic data indicate distinct protofilament structures. Scale

bar = 50 nm. The emission intensity of ThT staining of type A is
> 40-fold higher than that observed for types B and D. T
m
app
values
represent thermal melting midpoints during 90 °C
Æh
)1
temperature
ramping of 25 mgÆL
)1
fibrils in 25 mM glycine ⁄ HCl (pH 2.67). The
CD spectrum shows the reproducible unique fingerprint features of
fibrils after sonication that can be used to distinguish between the
types [27,43,56].
The structural ambiguity of glucagon amyloids J. S. Pedersen et al.
4592 FEBS Journal 277 (2010) 4591–4601 ª 2010 The Authors Journal compilation ª 2010 FEBS
molecular packing of the prion domain into the amy-
loid structure [32]. However, the discovery of self-
propagating variants of insulin [33], b
2
-microglobulin
[34], Ab [35] and glucagon [27] fibrils demonstrated
that strain behavior was not limited to proteins with a
prion-like sequence. Recently it has been demonstrated
that even small peptides, including the amylin frag-
ment SNNFGAILSS, have the ability to form amyloid
with both parallel and antiparallel b-sheet structures
[36,37] depending on the structure of the seeds. The
peptide GNNQQNY(7-13) forms two different types

of quasifibrillar crystal [38] and three different types of
amyloid according to solid-state NMR [39] that differ
from one another in subtle but distinct ways, such as
variations in the mobility of the aromatic Tyr ring.
There is a growing number of similar observations
from other peptide systems [40,41]. Remarkably, the
prevailing structure of insulin fibrils formed during
agitation appears to vary randomly between two
optically distinct polymorphisms [42], indicating that
indeterminism in early nucleation events dictates the
final fibril structure. We have previously demon-
strated that glucagon is able to form at least five
different fibrillar structures that can be propagated
by seeding in a strain-specific manner [23,27,43,44].
Each type can be identified by a unique combination
of variable characteristics, including thioflavin T
(ThT) staining, CD spectrum fingerprints, thermosta-
bility and morphology in EM (see Fig. 1). Type A,
B
agitated
,B
unagitated
all form in the same 50 mm gly-
cine ⁄ HCl buffer at pH 2.5, whereas types D and S
only accumulate when $ 200 mm Cl
)
and 1 mm
SO
2À
4

are added, respectively. Type A forms at high
glucagon concentrations (> 1 gÆL
)1
), whereas the
other types form at low concentrations (< 0.5 gÆL
)1
).
We have recently suggested that the prevalence of
different glucagon fibril structures is the result of a
multitude of aggregation pathways, where even small
shifts in environmental conditions can impact the
outcome of the struggle for monomers between sev-
eral types of fibril due to modulation of their nucle-
ation and exponential growth rates [23]. Interestingly,
another class of proteins for which environmental
conditions are critical in defining their structure is
the group of intrinsically disordered proteins that are
designed to show great conformational flexibility,
allowing them to interact with multiple binding
partners that can often induce different types of
structure [45,46]. In these cases, however, the poly-
morphism has been systematically optimized to facili-
tate heterogeneous contacts with different proteins
rather than the homogeneous assemblies illustrated
by the amyloid folds.
Breaking or branching fibril structures
allow exponential growth
When glucagon powder is dissolved in acidic buffer,
the fibril formation follows a highly reproducible sig-
moidal curve [47]. Sigmoidal curves are observed in a

number of biological systems, such as during exponen-
tial growth of bacteria in a flask or exponential ampli-
fication of DNA in PCR. Despite the obvious
similarities, a common misconception (as reviewed by
Roberts [48]) is that the lag time before the detection
of fibrils can be taken as a direct indicator of the pro-
pensity to slowly form a stable nucleus. For glucagon,
this is illustrated by simple seeding experiments, which
clearly demonstrate that the exponential growth phase
extends through the apparent lag all the way to the
beginning of the experiment [23] (Fig. 2C), which
means that the observed lag time depends inversely on
the growth rate of fibrils, as well as the nucleation
rate. A compilation of kinetic data from several pro-
teins shows a clear inverse correlation between the
apparent lag time and growth rates [49,50], suggesting
that fibril nucleation may generally take place through-
out the apparent lag [51], but exponential amplification
from early nucleation events will lessen the effect of
later nucleation events. The linear nature of fibrils
implies that they grow linearly by the addition of pro-
tein molecules to their ends [52]. Exponential growth
can be attributed to the presence of secondary path-
ways, which continuously increase the number of fibril
ends in proportion to the fibril mass present [51].
Recently, we have demonstrated using total internal
reflection fluorescence microscopy that a specific type
of glucagon fibril, which we refer to as type B
unagitated
[23], grows by branching under unagitated conditions

[53] (Fig. 2A). Branching in these fibrils is also
observed directly in high-resolution EM images [44],
which demonstrate that the fibrils consist of two or
more protofilaments that twist regularly. We speculate
that the twists may be necessary for fibrils to send out
branches or that cavities created on the surface of
these structures catalyze the formation of new fibril
nuclei. Fibrillogenesis of glucagon as well as a number
of other proteins, including insulin [54], Ab
1–40
[35]
and prions [55], can be greatly accelerated by agitation.
In the case of glucagon, clues to the mechanism of this
acceleration can be observed in the morphology of the
resulting fibrils, with agitation generally producing
type B
agitated
fibrils, which are short, nontwisted and
nonbranched parallel bundles of two or more filaments
[27]. We speculate that the type B
agitated
fibrils are brit-
tle fibrils that readily break during agitation, thereby
doubling the number of fibril ends that can accept
J. S. Pedersen et al. The structural ambiguity of glucagon amyloids
FEBS Journal 277 (2010) 4591–4601 ª 2010 The Authors Journal compilation ª 2010 FEBS 4593
monomers (Fig. 2B) and providing them with a selec-
tive growth advantage over type B
unagitated
fibrils under

agitated conditions [23]. The two types of fibril have
very similar CD spectra with an unusual positive peak
around 203 nm (Fig. 1) and two distinct b-sheet
peaks in FTIR spectra and a shoulder at 1664 cm
)1
,
indicating the presence of b-turns, suggesting that the
molecular packing could be very similar [27,56]. Agita-
tion-dependent molecular-level polymorphisms have
also been reported for Ab
1–40
and insulin fibrils
[35,57]. Moreover, different types of prion fibril form
under shaking and rotating conditions, indicating that
the mode of agitation can also influence the prevailing
pathway of fibrillogenesis [58]. Quiescent (unagitated)
and agitated forms of Ab
1–40
fibrils exhibit twisted
and striated ribbon morphologies similar to the type
B
unagitated
and B
agitated
glucagon fibrils, respectively.
With rounds of seeded growth it has been possible to
generate homogeneous samples of quiescent and agi-
tated Ab
1–40
fibrils that allowed a solid-state NMR

study of underlying structural differences [59]. The
structures reveal that the secondary structure of Ab
1–40
in the two forms is very similar, but that the quiescent
form has a triangular cross-section with three protofil-
aments with a narrow cavity in the middle, whereas
the agitated striated ribbon form consists of two proto-
filaments with a tight interaction between the flat sur-
faces between them. It has recently been demonstrated
that specific types of the Ab
1–40
fibril can also grow by
branching [60]. Investigations on the similarities and
differences between the type B
unagitated
and B
agitated
glucagon fibril forms are currently being conducted.
Transient off-pathway formation of
monofilament type A fibrils allows
growth of type B fibrils at high
glucagon concentrations
Based on time-lapse EM and AFM studies alone, it has
been proposed that formation of the complex
multifilament structures of mature fibrils may proceed
by lateral assembly of preformed protofilaments or pro-
tofibrils [29,61,62]. Even though EM and AFM provide
high-quality information about the fibril structures
Fig. 2. Secondary pathways in glucagon fibrillogenesis result in exponential growth of mature fibrils. (A) Under unagitated conditions, TIRF
microscopy directly demonstrates that type B

unagitated
fibrils increase the number of fibril ends by branching, leading to exponential growth.
Adapted from [53]. (B) Under agitated conditions, type B
agitated
fibrils have selective growth advantage because they continuously break,
thereby exposing new fibril ends that adsorb monomeric glucagon [27]. (C) Seed extension kinetics of 1 gÆL
)1
glucagon in 50 mM
glycine ⁄ HCl pH 2.5 with agitation confirm that type B
agitated
fibrils grow exponentially, and indicate that a small fraction ($ 1in10
5
)of
glucagon molecules initiate spontaneous nucleation as soon as glucagon is dissolved under the given conditions [23].
The structural ambiguity of glucagon amyloids J. S. Pedersen et al.
4594 FEBS Journal 277 (2010) 4591–4601 ª 2010 The Authors Journal compilation ª 2010 FEBS
formed, many images need to be analyzed to quantify
the amounts of various structures formed. Moreover,
the images do not provide information about differ-
ences in the molecular packing of fibrils and it is virtu-
ally impossible using these techniques alone to quantify
the fraction of the protein that has become converted
to fibrils at the given time point. The hierarchical
build-up model seems to conflict with recent data that
suggest that different types of glucagon fibril grow
exponentially via their own distinct pathways into
structurally distinct entities [23,27,43,44,53,56]. Several
studies have revealed that the formation of monofila-
ment type A fibrils (Fig. 1), which appear as single fila-
ments in EM and AFM, occurs at glucagon

concentrations above 1 gÆL
)1
[27,44,63]. When agitated
at 1 or 2 gÆL
)1
, transient formation of type A fibrils
can be observed as a peak in ThT emission, which dis-
appears as type B
agitated
fibrils form [27]. However,
under unagitated conditions at higher concentrations,
the type A fibrils form a thick gel [47] that appears to
be stable for longer periods, which makes it possible to
study the properties of these otherwise metastable
fibrils [44]. A recent study summarized the evidence for
structural differences between type A and B
unagitated
[56]: unlike type B fibrils, type A fibrils have an unusu-
ally strong b-sheet CD spectrum (Fig. 1) and an FTIR
spectrum with only one b-sheet peak. Linear dichroism
of aligned fibrils indicates that type B
unagitated
fibrils are
less ordered than type A [56]. X-ray diffraction patterns
reveal that both types exhibit the classical 4.76 A
˚
meridional reflection typical for amyloid-like structures
[18], but whereas type B
unagitated
only contains the clas-

sical 9.8 A
˚
equatorial reflection, type A fibrils exhibit a
number of periodic reflections similar to those of a cyl-
inder with well-defined edges. This suggests that the
simple structure of type A fibrils can be aligned more
orderly than the branched type B
unagitated
fibrils [56].
Limited proteolysis results in the release of a different
spectrum of peptides, further substantiating the struc-
tural differences between the two types of fibril [56].
The two fibril types also differ in terms of the mecha-
nisms leading to their formation, as evident from
kinetic cross-seeding experiments: Fractionated seeds of
both type A and B
unagitated
fibrils can grow exponen-
tially at low concentrations, but type B
unagitated
fibrils
have a faster exponential growth (a more shallow slope
Fig. 3. A proposed mechanism for the
conversion of type A fibrils into type B. (A)
Seeding experiments demonstrate that
seeds of type A fibrils can grow at both high
and low glucagon concentrations. In
contrast, seeds of type B
unagitated
fibrils

grow exponentially only at low glucagon
concentrations, possibly due to inhibition of
either elongation or branching by a-helical
trimers at high concentrations [44]. (B) Once
type A fibrils have consumed > 95% of the
monomers, the concentration is low enough
to allow exponential growth of type
B
unagitated
fibrils. A rapid subsequent
equilibrium between monomers and the
relatively unstable type A fibrils ensures that
glucagon monomer concentrations are
maintained at low enough levels to support
the growth of type B
unagitated
fibrils. Data
for the graph were taken from the recent
SAXS study [63].
J. S. Pedersen et al. The structural ambiguity of glucagon amyloids
FEBS Journal 277 (2010) 4591–4601 ª 2010 The Authors Journal compilation ª 2010 FEBS 4595
on a lag time versus log[seed] plot) [44]. In contrast,
only type A fibrils seed exponential growth at high con-
centrations, possibly because either the branching or
elongation of type B
unagitated
seeds is inhibited by the
a-helical trimers that form in equilibrium with mono-
mers at these concentrations [64] (Fig. 3A). Consistent
with this, the apparent lag time for the formation of

type B
unagitated
fibrils actually increases with increasing
glucagon concentrations above 0.3 gÆL
)1
[44]. All of the
abovementioned differences make it difficult to propose
that type B
agitated
fibrils could be assembled by simple
lateral associations of several type A protofibrils – the
properties of the structures are simply too different. We
therefore hypothesize that conversion from type A to
mature type B
unagitated
fibrils over time could occur by
gradual shedding of monomers from unstable type A
fibrils that subsequently adsorb to the more stable
exponentially growing type B
agitated
fibril structure.
Data from thermal melting suggest that type A fibrils
are relatively unstable compared with type B
agitated
fibrils, with apparent thermal melting midpoints (T
app
m
)
of < 32 and 55 °C, respectively [27]. Moreover, linear
extrapolation of urea dissociation kinetics indicates

that type A fibrils have a much faster dissociation rate
of $ 0.69 h
)1
compared with the 0.03 h
)1
observed for
type B
agitated
fibrils [27]. This corresponds to a half-life
of only 1 h if type A fibrils were diluted infinitely in
buffer. Proteolysis with pepsin, which continuously
degrades flexible monomers more readily than fibrils,
shows nearly the same value [27]. Recent developments
in small angle X-ray scattering (SAXS) allows noninva-
sive quantitative analysis of the relative amounts of
fibrils consisting of single (type A) and multiple proto-
filaments (type B
unagitated
) (green and orange curves in
Fig. 3B, respectively) [63], which is very difficult if not
impossible to achieve using combinations of ThT and
Trp fluorescence alone. Data from the SAXS study
indicate that type A fibrils grow until they have
consumed nearly all of the glucagon (blue curve
in Fig. 3B) before the exponential growth of type
B
unagitated
fibrils reaches detectable levels. Interestingly,
the SAXS data show that the lag time for the forma-
tion of type B

unagitated
increases from 18 h (5 gÆL
)1
)to
35 h (10 gÆL
)1
) [63], indicating that the growth of type
B
unagitated
depends on the remaining nonfibrillated
glucagon concentration rather than on the amount of
type A fibrils formed before them [63]. This is inconsis-
tent with type A being a structural prerequisite for the
formation of type B
unagitated
fibrils, but consistent with
the quantitative cross-seeding data [44] that suggests
type B
unagitated
fibrils are unable to grow before mono-
mer concentrations are sufficiently low (i.e. due to the
formation of type A). Because type A fibrils have a half-
life of only 1 h, their shedding of monomers apparently
keeps concentrations at a sufficient level to facilitate
rapid growth of type B
unagitated
fibrils. Thus, it appears
that the transition from type A to B fibrils probably
occurs via shedding and adsorption of monomers.
It has been reported that multiple distinct assembly

pathways may be responsible for the formation of pro-
tofibrillar and mature fibrillar structures of Ab [65],
b
2
-microglobulin [66] and Sup35NM [67]. Clearly,
future studies should include kinetics experiments and
structural data before concluding that fibrils form via
a hierarchical build-up mechanism [68]. Nevertheless, it
is unlikely that there will be a single unifying mecha-
nism for the build-up of fibrils from its constituents.
The diversity of possible interactions due to different
protein sequences is simply too great [29]. There are
cases where preformed oligomers can be demonstrated
to be incorporated directly into the fibrils [69], and
kinetic data from SAXS also support that insulin
fibrils could be built from preformed oligomeric build-
ing blocks [70], although the mechanism that leads to
a lag time before the accumulation of the building
block has not yet been described in detail. As a further
example, our SAXS studies of the fibrillation of a-syn-
uclein under agitated conditions identify three species,
namely a monomer ⁄ dimer state, an oligomer with
a central channel and an extended fibril (L. Giehm,
D. Svergun, D. E. Otzen and B. Vestergaard, submit-
ted). Structurally and mechanistically this oligomer
appears to be a direct precursor to the fibril.
Charge neutrality in type B fibrils
At the acidic pH used for the fibrillogenesis of gluca-
gon, histidine residue 1 (His1), the three aspartic acid
residues (Asp9, Asp15 and Asp21) and the C-terminus

exist mostly in the protonated state. This means that
glucagon has a net charge of +5 (N-terminus, His1,
Lys12, Arg17 and Arg18). If left unshielded, this would
lead to high static repulsion, which is irreconcilable
with the close packing of glucagon molecules that
occurs in fibrils. There are two possible mechanisms
that would allow glucagon fibrils to exist at low pH:
either the pK
a
values are shifted so that glucagon mole-
cules in fibrils lose some of these charges or counter
ions from the solution shield the positive charges. The
protonation state of type B
agitated
glucagon fibrils has
been investigated by isothermic titration calorimetry
(ITC) during extension of seeds [71]. Using a series of
buffers with different protonation enthalpies, it was
possible to measure how many protons were exchanged
with the buffer upon incorporation of a glucagon mole-
cule into a seed. The data obtained are consistent with
The structural ambiguity of glucagon amyloids J. S. Pedersen et al.
4596 FEBS Journal 277 (2010) 4591–4601 ª 2010 The Authors Journal compilation ª 2010 FEBS
the release of five protons upon monomer addition to a
fibril end, indicating that glucagon is charge neutral in
the type B
agitated
fibrillated state [71] (Fig. 4). Consis-
tently, the stability of type B
agitated

depends on pH,
with fibrils dissociating instantly at pH 1.1 and T
app
m
increasing from 40 °C at pH 2.1 to 61 °C at pH 3.2
[27]. Glucagon can also form fibrils in glycine ⁄ NaOH
buffer at pH 9.5 [72], where monomeric glucagon is
expected to have a net charge of $ )1 [73], and accord-
ing to data from ITC experiments, the molecules also
become charge neutral when incorporated into these
fibrils [71]. Because of the charge neutrality of fibrils at
both high and low pH, it is conceivable that the type of
molecular packing of glucagon in fibrils formed at these
very different pH values could be identical. The obser-
vation that seeds of fibrils formed at pH 9.5 can grow
exponentially at pH 2.5, with kinetics that are virtually
superimposable to kinetics of type B
agitated
seeding sug-
gests that type B
agitated
fibrils can also form at pH 9.5
(J. S. Pedersen, unpublished data).
Shielding of charges by ions allows
fibrils with alternative molecular
structures
Because of the +5 charge on monomeric glucagon mol-
ecules at acidic pH, shielding of charges by anions can
increase the rate of fibril formation by relieving the
charge repulsion between monomers [43]. However,

salts appear to favor the growth of fibrils with alterna-
tive properties: in the presence of 150–250 mm Cl
)
, type
D fibrils appear to have a selective growth advantage
over type B
agitated
fibrils, even under agitated conditions
[27], possibly because the salts also stabilize type B
agitated
fibrils, making them less prone to break [27]. The diva-
lent anion SO
2À
4
is 125-fold more stabilizing than Cl
)
for
type S fibrils, and every 10-fold increase in salt concen-
tration increases T
app
m
with 22 °C for, implying that salts
are critical to stability of Type S fibrils [43]. This
explains why adding as little as 1 mm SO
2À
4
gives a selec-
tive growth advantage to type S fibrils. We have previ-
ously speculated that the negative charges on SO
2À

4
could allow packing of positively charged glucagon mol-
ecules into fibrils, as well as lateral associations between
several positively charged fibrils or protofilaments
thereby increasing stability [43].
A comparison of the X-ray diffraction patterns
reveals slight differences in the interstrand distance of
type B, S and D fibrils with meridionals at 4.7, 4.8 and
4.9 A
˚
, respectively [73]. Each fibril type also has their
own signature of equatorial reflections. Moreover,
Trp25 appears significantly more exposed to acrylamide
quenching in type B
agitated
fibrils compared with type D
and S [73]. Convincing evidence for structural differ-
ences between the three types of fibril also comes from
cross-seeding experiments under identical conditions
(e.g. 1 gÆL
)1
monomeric glucagon in 50 mm glycine pH
2.5), which lead to propagation of the structure of the
seed [27,43]. We have used ITC to extend this charac-
terization with a thermodynamic comparison of type
B
agitated
, S and D fibrils [73]. By measuring the enthalpy
change, DH, for seeded fibril elongation at a series of
temperatures, it is possible to estimate the change in

heat capacity for fibril formation (DC
p
) for the three
fibril types [74]. Remarkably, the DC
p
values for the
fibril extension of the three types are significantly dif-
ferent, with positive values for type D and negative val-
ues for type S (Fig. 5). For type B
agitated
, the enthalpy
Fig. 4. Data from ITC experiments during seed extension indicate
that each monomer releases five protons at pH 2.5 and takes up
one proton at pH 9.5 upon addition to a type B
agitated
fibril end [71].
Fig. 5. Thermodynamic analysis of fibrillar polymorphism. Enthalpy
change during extension of various types of glucagon fibril as a
function of temperature. The slope of these curves corresponds to
DC
p
values, which are strikingly different for the three types of
fibril. Adapted from [73].
J. S. Pedersen et al. The structural ambiguity of glucagon amyloids
FEBS Journal 277 (2010) 4591–4601 ª 2010 The Authors Journal compilation ª 2010 FEBS 4597
is dominated by buffer deprotonation, making the
intrinsic DC
p
essentially zero. It is difficult to identify a
simple structural basis for this remarkable variation in

DC
p
values. Clearly the predicted change in solvent
accessible area, which correlates strongly with DC
p
for
globular proteins [75], is not a useful predictor of fibril-
lar DC
p
. It is possible that strong backbone interactions
lead to the unfavorable burial of polar side residues,
water and ⁄ or charged groups, which can all have major
influence on the change in DC
p
.
Future perspectives – toxicity of
alternative protein folds
So far we have demonstrated that glucagon is able to
form at least five types of amyloid fibril that appear to
differ at the level of their molecular packing of gluca-
gon. Based on some of the structures observed in EM,
it is very likely that several other types of fibril with
unique properties remain to be discovered. Judging by
seed extension kinetics and the overall sigmoidal shape
of the growth curves, it appears logical to assume that
they all grow exponentially by monomer addition from
rare thermodynamic nuclei that start to form when
glucagon is dissolved (Fig. 6).
In the current research on protein aggregation and
amyloid formation, interest in prefibrillar intermediate

structures and other oligomers is growing, given that
the toxicity of these species appears to surpass that of
mature fibrils [76]. It is possible that the toxicity of
aggregates is simply correlated directly with the surface
to mass ratio, implying that smaller structures, which
have a high surface to mass ratio, are more toxic than
large aggregates, which have a small surface to mass
ratio. However, it is becoming evident that specific
folds in oligomers can be significantly more toxic than
others [77]. It has been reported that mishandling of
glucagon solutions of > 2 gÆL
)1
leads to the formation
of toxic aggregates [78]. Moreover, a recent report
comparing the toxicity of amyloid structures of several
protein hormones indicated that fibril preparations
formed during a 14 day incubation at 37 °C with slight
agitation of 2 gÆL
)1
glucagon in the presence of
0.4 mm low relative molecular mass heparin and 5%
d-mannitol at pH 5.5 are particularly toxic, resulting
in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide reduction exceeding that caused by Ab
1–42
and Ab
1–40
aggregates [16]. Unfortunately, the study
did not reveal what specific type(s) of glucagon aggre-
gate(s) cause toxicity. Several studies have been aimed

at characterizing prefibrillar intermediate species of
glucagon and oligomeric structures have been reported
in AFM studies [61,62]. However, according to data
from field flow fractionation [79], NMR [80], SAXS
[63] and dynamic light scattering [44], the benign
a-helical trimer, which is in rapid equilibrium with
monomers [44,64,81] and crystallizes readily [14,82], is
the only oligomeric structure that forms at detectable
levels at pH 2.5. It is possible that a shift to pH 5.5,
where the molecules have an average charge of +1,
could allow glucagon to form more stable toxic oligo-
meric species. Another possibility is that the aggre-
gated species responsible for toxicity is a type of fibril,
which raises the question of what type of fibril is
responsible for toxicity. With its fibrillation mecha-
nisms and fibrillar polymorphisms being so well under-
stood, glucagon appears to be an excellent model
system for future studies to further our under-
standing of the relationship between protein aggregate
structures and toxicity.
Acknowledgements
We gratefully acknowledge Dr Hans Aage Hjuler and
coworkers at Novo Nordisk A⁄ S for extensive fund-
ing over the years as well as generously providing
unlimited amounts of the highest possible quality of
glucagon samples. We are also grateful to Drs Chris-
tian Rischel, Peter Westh and James Flink for fruitful
collaborations and stimulating discussions. JSP is sup-
ported by the Carlsberg Research Foundation. DEO
acknowledges support from the Danish Research

Fig. 6. Summary of the five different types of glucagon fibril inves-
tigated in detail and the proposed nucleation-dependent pathways
that lead to their formation. On each pathway monomers are in
equilibrium with individual thermodynamic nuclei, which are the
most unstable transition state between monomers and fibrils.
According to the monomer concentration dependence of seeded
fibril elongation, all fibril types grow by monomer addition
[27,43,44]. The equilibrium between a-helical trimers and
monomers inhibits exponential growth and ⁄ or nucleation of type
B
unagitated
. It is possible that the growth of other types of mature
fibril could similarly be inhibited by a-helical trimers.
The structural ambiguity of glucagon amyloids J. S. Pedersen et al.
4598 FEBS Journal 277 (2010) 4591–4601 ª 2010 The Authors Journal compilation ª 2010 FEBS
Foundation (inSPIN). CBA is supported by a postdoc-
toral fellowship financed by The Benzon Foundation
and Novo Nordisk.
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