Molecular basis for insulin fibril assembly.
ABSTRACT In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of full-length insulin in a molar ratio-dependent manner, suggesting that this segment is central to the cross-beta spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 A resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated beta-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies.
- SourceAvailable from: Santiago Schnell[Show abstract] [Hide abstract]
ABSTRACT: We propose three new reaction mechanisms for competitive inhibition of protein aggregation for the two-step model of protein aggregation. The first mechanism is characterized by the inhibition of native protein, the second is characterized by the inhibition of aggregation-prone protein and the third mechanism is characterized by the mixed inhibition of native and aggregation-prone proteins. Rate equations are derived for these mechanisms, and a method is described for plotting kinetic results to distinguish these three types of inhibitors. The derived rate equations provide a simple way of estimating the inhibition constant of native or aggregation-prone protein inhibitors in protein aggregation. The new approach is used to estimate the inhibition constants of different peptide inhibitors of insulin aggregation.Biophysical Chemistry 09/2014; · 2.32 Impact Factor
Chapter: Stability of Amyloid Oligomers[Show abstract] [Hide abstract]
ABSTRACT: Molecular simulations are now commonly used to complement experimental techniques in investigating amyloids and their role in human diseases. In this chapter, we will summarize techniques and approaches often used in amyloid simulations and will present recent success stories. Our examples will be focused on lessons learned from molecular dynamics simulations in aqueous environments that start from preformed aggregates. These studies explore the limitations that arise from the choice of force field, the role of mutations in the growth of amyloid aggregates, segmental polymorphism, and the importance of cross-seeding. Furthermore, they give evidence for potential toxicity mechanisms. We finally discuss the role of molecular simulations in the search for aggregation inhibitorsAdvances in Protein Chemistry and Structural Biology, Edited by Tatyana Karabencheva-Christova, 09/2014: chapter CHAPTER FOUR: pages 113-141; Academic Press.
- [Show abstract] [Hide abstract]
ABSTRACT: Herein we report that protein fibrils formed from aggregated proteins, so called amyloid fibrils, serve as an excellent dispersing agent for hydrophobic oligothiophenes such as α-sexithiophene (6T). Furthermore, the protein fibrils are capable of orienting 6T along the fibril long axis, as demonstrated by flow-aligned linear dichroism spectroscopy and polarized fluorescence microscopy. The materials are prepared by solid state mixing of 6T with a protein capable of self-assembly. This results in a water soluble composite material that upon heating in aqueous acid undergoes self-assembly into protein fibrils non-covalently functionalized with 6T, with a typical diameter of 5-10 nm and lengths in the micrometre range. The resulting aqueous fibril dispersions are a readily available source of oligothiophenes that can be processed from aqueous solvent, and we demonstrate the fabrication of macroscopic structures consisting of aligned 6T functionalized protein fibrils. Due to the fibril induced ordering of 6T these structures exhibit polarized light emission.Journal of materials chemistry C. 07/2014; 2:7811-7822.
Molecular basis for insulin fibril assembly
Magdalena I. Ivanovaa, Stuart A. Sieversa, Michael R. Sawayaa, Joseph S. Wallb, and David Eisenberga,1
aHoward Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, Los Angeles CA 90095-1570; andbBiology Department, Brookhaven
National Laboratory, Upton, NY 11973-5000
Contributed by David S. Eisenberg, September 11, 2009 (sent for review August 7, 2009)
In the rare medical condition termed injection amyloidosis, extra-
cellular fibrils of insulin are observed. We found that the segment
of the insulin B-chain with sequence LVEALYL is the smallest
segment that both nucleates and inhibits the fibrillation of full-
length insulin in a molar ratio–dependent manner, suggesting that
this segment is central to the cross-? spine of the insulin fibril. In
isolation from the rest of the protein, LVEALYL forms microcrys-
we determined to 1 Å resolution. The LVEALYL segments are
stacked into pairs of tightly interdigitated ?-sheets, each pair
displaying the dry steric zipper interface typical of amyloid-like
fibrils. This structure leads to a model for fibrils of human insulin
consistent with electron microscopic, x-ray fiber diffraction, and
amyloid ? fibril structure
full-length insulin molecules are found in fibrillar form at the site
of frequent insulin injections (2–4). These insulin fibrils formed
in vivo display the defining characteristics of amyloid aggregates
(5), including binding the dye Congo red with ‘‘apple-green’’
birefringence, an elongated, unbranched fibrillar morphology
(4), nucleation-dependent polymerization, and the cross-? x-ray
with Parkinson’s disease have been found to display an autoim-
mune response to insulin oligomers and fibrils (9), possibly
indicating the presence of insulin aggregates in this disease as
well. Insulin also forms amyloid-like fibrils in vitro, which are
promoted by elevated temperatures, low pH, and increased ionic
strength (4, 10). In addition, insulin fibril formation has been a
limiting factor in long-term storage of insulin for treatment of
diabetes. Thus, better understanding of insulin fibrillation could
lead to safer handling and more cost-effective storage of insulin.
Because insulin offers the structural simplicity of two short
polypeptide chains constrained by one intramolecular and two
fibrils have been numerous. Upon fibrillation, the molecule of
insulin undergoes structural changes from a predominantly
?-helical state to a ?-sheet rich conformation. The fibrillar
?-sheets have been described as either parallel (11–13) or
antiparallel (14–16). An early model was based on the crystal
packing of a despentapeptide insulin molecule (with residues
B26–B30 removed) (4, 17). More recently, Jimenez et al. (18)
assembly the ?-helical insulin molecules undergo conforma-
from x-ray solution scattering and ab initio modeling, Vester-
gaard et al. (19) proposed that insulin fibrils are formed by
primarily ?-helical oligomers. The first atomic-level view of the
interactions between segments of insulin which may be part of
fibrillar spine came from single crystal structures of the fibril-
forming peptide segments LYQLEN (residues A13–A18) and
VEALYL (residues B12–B17) (16).
Previous biophysical studies suggest that the B chain, or a
segment of it, may be the primary determinant of insulin
fibrillation. For example, equimolar amounts of the peptide
RRRRRRLVEALYLV (containing residues B11-B17 of the B
ne of the roughly 25 disorders categorized as amyloid
diseases (1) is injection amyloidosis. In this rare condition,
chain) can attenuate insulin fibrillation (20). In addition, the
point mutations H10D and L17Q in the B chain of insulin
prolong the lag phase of insulin fibrillation, further supporting
the importance of this segment in fibril formation (21). Also,
exposing this fibril-prone segment by truncating the C-terminal
five residues of the B chain increases the propensity of insulin for
fibril formation (4, 17).
Other studies have shown that the A chain also contributes to
insulin fibrillation. Both A chain and B chain can form fibrils on
their own (22, 23), and seeds of A chain or B chain can nucleate
the fibrillation of full length insulin (22). In addition, it was
reported that segments as short as six residues from either A
chain (residues A13–A18) or B chain (residues B12–B17) can
form fibrils by themselves (24). The same segments from A chain
(residues A13–A19) and B chain (residues B9–B19) were found
to be protected against hydrogen exchange when insulin was
on these findings, it seems that a segment from the A chain may
also be involved in the formation of the spine of insulin fibrils.
Much evidence has accumulated in support of the view that
only specific segments of amyloidogenic proteins form the spine
of amyloid fibrils. Peptide segments, as short as three to four
residues, can form fibrils by themselves in vitro (26–30). Fur-
thermore, these segments from the spine of the fibril often act
as inhibitors of fibrillation of their parent proteins. For example,
(residues 16–20) and IAPP (residues 22–27) can attenuate fibril
formation of the full-length protein (31–35).
Our work suggests that the B-chain segment LVEALYL is the
main contributor to the spine formation of fibrils of full-length
insulin. The crystal structure of the segment LVEALYL pro-
vides a molecular view of the structural organization of the spine
of insulin fibrils and was used to build a model of fibrils of the
full-length protein. The model is supported by the x-ray fiber
diffraction of insulin fibrils and scanning-transmission electron
microscopy (STEM) analysis of the morphology of insulin fibrils.
Insulin Segments Form Fibrils and Alter Lag Time of Insulin Fibril
when co-incubated with full-length protein are likely to partic-
ipate in the spine of full-length fibrils. Based on previous studies
(24), there are two segments of insulin that form fibrils in
isolation from the rest of the protein; these are VEALYL from
the B chain and LYQLEN from the A chain.
To ascertain whether either or both of these segments form
the spine of insulin fibrils, we studied their effects on the rate of
Author contributions: M.I.I., S.A.S., and D.E. designed research; M.I.I., S.A.S., and M.R.S.
performed research; M.I.I., M.R.S., and J.S.W. contributed new reagents/analytic tools;
M.I.I., S.A.S., and M.R.S. analyzed data; and M.I.I., S.A.S., M.R.S., and D.E. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The coordinates of the crystal structure of LVEALYL have been deposited
with the Protein Data Bank, www.pdb.org (PDB ID code 3HYD; structure factors PDB ID
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
November 10, 2009 ?
vol. 106 ?
fibrillation of full-length insulin. Both of these segments are
located between the two interchain disulfide bonds of full-length
insulin (Fig. 1A). In our first experiments, we tested the effect on
insulin fibril formation of nine six-residue segments with se-
quences spanning the A and B chain segments between the two
interchain disulfide bonds of insulin [Supporting Information
(SI) Table S1]. None of the nine six-residue segments affected
the fibrillation of full-length insulin. These results suggest that
the spine of insulin fibrils is longer than six residues.
Searching for the smallest segment of insulin that affects the
kinetics of fibrillation of full-length insulin, we synthesized two
LVEALYLV (B chain) (Fig. 1A). Both SLYQLENY and
LVEALYLV form fibrils in solution (Fig. 1D). As seen in Fig.
1B, the A chain segment SLYQLENY does not affect insulin
fibrillation. In contrast, the B chain segment LVEALYLV slows
insulin fibrillation when incubated in molar concentrations down
to 10 times less than that of insulin (Fig. 1C). The strongest
inhibitory effect of LVEALYLV is observed when incubated in
equimolar concentrations with insulin. The lag time of fibril
formation with LVEALYLV at a 1:1 molar ratio is more than 10
fibrils in electron micrographs of the sample of insulin with
LVEALYLV (1:1) compared with the densely packed fibrils
observed that LVEALYLV nucleated fibril formation when
incubated in molar concentrations 40 times less than insulin (Fig.
1C). Thus, depending on the concentrations used, LVEALYLV
either inhibits or nucleates fibril formation.
Given that the six-residue segments do not affect the rate of
insulin fibril formation and that the eight-residue segment
LVEALYLV does, we wanted to know whether either of the two
seven-residue peptides within LVEALYLV affected fibril for-
mation. The two seven-residue peptides LVEALYL and
VEALYLV formed fibrillar aggregates (Fig. 2A and Fig. S1A).
However, only LVEALYL inhibited fibril formation of full-
length insulin (Fig. 2C and Fig. S1B), and there were only sparse
(Fig. 2B). Also, depending on its molar ratio relative to that of
fibril formation (Fig. 2C).
Structure of LVEALYL. The segment LVEALYL forms microcrys-
tals diffracting to 1 Å resolution, and we were able to determine
SLYQLENY of the A chain is dark red. Segment LVEALYLV of the B chain is dark blue. Disulfide bonds are colored in yellow. (B) SLYQLENY of the A chain, when
added to the reaction mixture, does not affect the rate of full-length insulin fibril formation. Fibrillation was monitored as a function of time by measuring ThT
fluorescence. Full-length insulin starts to form fibrils after 8–10 h, and its conversion is complete in ?20 h. (C) Fibrillation assay showing LVEALYLV from the B
at concentrations 25–40 times less than the concentration of insulin. Note that neither SLYQLENY (A chain) nor LVEALYLV (B chain) fluoresce in the presence
of ThT. All points represent the mean value of at least four replicates, with error bars representing the SD. (D) Electron micrographs showing that SLYQLENY
and LVEALYL aggregates are fibrillar in morphology (E). LVEALYLV in equimolar ratios inhibits insulin fibril formation. Many fibrils were observed in the
micrograph of the sample of full-length insulin taken 48 h after the beginning of fibrillation assay (left). In contrast, there were only a few fibrils in the sample
of insulin incubated at equimolar ratio with LVEALYLV (right). (Scale bars, 400 nm.)
An eight-residue segment from the insulin B chain accelerates and inhibits insulin fibril formation. (A) Amino acid sequence of insulin. Segment
of insulin fibril formation. (A) Electron micrograph of fibrillar aggregates of
B-chain LVEALYL. (B) Electron micrograph of sample taken from equimolar
solution of insulin and LVEALYL after 48 h from beginning of assay. Note that
there are only sparse fibril-like aggregates. (C) Fibrillation assay showing that
B-chain LVEALYL accelerates insulin fibril formation when added to the
reaction mixture at low concentrations, but inhibits insulin fibril formation at
higher concentrations. (Scale bars, 400 nm.)
LVEALYL from B chain is the smallest segment that can alter the rate
Ivanova et al.PNAS ?
November 10, 2009 ?
vol. 106 ?
no. 45 ?
its fibril-like structure. Extended strands of LVEALYL pack in
register into parallel ?-sheets, which run the entire length of the
needle crystal (Fig. 3A). Alternating side chains L, E, L, and L
of side chains extending from the second sheet intermesh with
those from the first, forming a dry, highly complementary
interface of the type termed ‘‘steric zipper’’ (16). Each pair of
sheets forming the dry interface packs against two other iden-
tical steric zippers, separated by wet interfaces containing six
water molecules (Fig. 3C). The dry interface of the LVEALYL
steric zipper buries a larger surface area and displays higher
surface complementarity than does the steric zipper of the
structure of VEALYL, which we determined earlier (16).
Mass per Unit Length of Insulin Fibrils. A measurement that tells
much about the molecular structure of a fibril is its mass per unit
length (MPL), which can be determined by STEM. The STEM
observations were made on insulin fibrils grown under the same
conditions used in the fibril kinetic assays. The most common
MPL values of the narrowest fibrils clustered around a mean of
2.85 ? 0.35 kDa/Å (Fig. 4A). This MPL value of 2.85 ? 0.35
kDa/Å corresponds well with the value of 2.47 kDa/Å expected
for two insulin molecules (Mr ? 2 ? 5808 Da) per 4.7 Å rise of
an amyloid fibril (2 ? 5.81 kDa/4.7Å ? 2.47 kDa/Å). Other
common MPL values clustered around two and three times this
value, suggesting that these fibrils consist of two and three of the
narrowest measured filaments. Also observed was a clustering
around an MPL value of about four times that of the narrowest
measured filament. As seen in the Fig. 4B, the morphologies of
cross-over distances. For example, the cross-over distances of
fibrils with MPL values of 2.85 kDa/Å and 5.43 kDa/Å is ?1,200
Å. This value was used in building our model of insulin fibrils
each layer of the steric zipper of the fibril spine is not only
separated by 4.7 Å from the layer below but is also rotated by
?0.71° ([4.7 Å/2400 Å] ? 360°).
here shows that the seven-residue B-chain segment LVEALYL
can either delay or accelerate insulin fibril formation in a molar
ratio–dependent manner. In equimolar ratios with insulin,
LVEALYL inhibits fibrillation, and in much lower concentra-
tions than insulin LVEALYL accelerates it. This dual mode of
action of the B-chain segment LVEALYL on insulin fibril
formation suggests that this segment is important for the for-
mation of the spine of the insulin fibrils. In contrast, segments
from the A chain lack this dual mode of action. The experiments
represented in Fig. 1B show that the eight-residue A-chain
segment SLYQLENY does not affect the rate of insulin fibril
formation. Apparently this segment, although bound to the
B-chain through two disulfide bonds, is peripheral to the spine.
In short, our experiments on fibrillation indicate that the spine
by LVEALYL molecules. Crystal needle length runs vertical in this orientation. (B) View down fibril axis showing one layer of interdigitated pair of LVEALYL
molecules, which interlock tightly to form the dry steric zipper interface. Pairs of extended ?-strands of LVEALYL are stacked in register upon each other, so this
figure may be thought of as a projection of two ?-sheets, each containing some 100,000 layers. Note that this dry steric zipper interface is devoid of water
molecules (shown in yellow). (C) Packing of LVEALYL molecules in crystal, viewed down fibril axis as in (B). Molecules forming the dry steric zipper interface are
to MPL value of 2.47 kDa/Å of the insulin fibril model shown in Fig. 5. The
insulin fibril model contains two molecules of insulin per 4.7-Å layer. MPL
values of the thicker fibrils correspond to fibrils with four, six, and eight
molecules of insulin per 4.7-Å layer. Histogram was produced by using a
binning window of 0.25 kDa/Å. (B) Electron micrographs of insulin fibrils
representing the MPL of the fibril populations shown in (A). Note that fibrils
suggesting that particles with larger MPL values comprise fibrils with the
smallest MPL value of 2.85 kDa/Å. (Scale bars, 100 nm.)
STEM measurements of MPL of insulin fibrils. (A) MPL value of the
www.pnas.org?cgi?doi?10.1073?pnas.0910080106 Ivanova et al.
of insulin fibrils includes the segment of the B chain between the
interchain disulfide bonds, and model building of the fibril was
based on this basic observation. In addition, the A chain is
covalently bound to the B chain via two interchain disulfide
bonds and contributes peripherally to the fibril. The crystal
structure of LVEALYL shows how the A chain can be accom-
modated at the periphery of the spine, as explained below.
Dual Action of LVEALYL on the Rate of Fibrillation. Our observation
that the segment LVEALYL is an effective inhibitor of insulin
fibrillation, and yet when present in low molar ratios to insulin
can accelerate fibril formation, has precedents. In two separate
studies, ?1-antichymotrypsin has been observed to both accel-
erate and inhibit ?-amyloid (A?) fibrillation. For example,
?1-antichymotrypsin, when incubated with A? peptides at ratios
lower than 1:100, accelerates A? fibrillation (36). However,
when the ?1-antichymotrypsin to A?1–40 ratio is raised to 1:10,
fibril formation is inhibited (37). Apoliprotein E4 (Apo E4) is
another example in which fibrillation of A? peptide is both
inhibited and accelerated. In contrast with LVEALYL and
?1-antichymotrypsin, the apo E4 dual effect on A? fibrillation is
reversed: Apo E4 inhibits fibrillation when incubated at con-
centrations lower than those that accelerate fibril formation (36,
38, 39). The dual effect of action of apo E4 and ?1-antichymo-
trypsin on fibrillation of A? was reported in separate studies.
This study shows that a short peptide segment can also nucleate
and inhibit fibril formation.
The molecular mechanism of the dual effect is unknown. One
possibility is that, at low concentrations, LVEALYL nucleates
the spine of full-length insulin fibrils more rapidly than do insulin
molecules. This is reasonable because the side chains of the
segment LVEALYL are exposed, and hence a small group of
molecules can interact with each other by intermeshing their side
chains, thereby forming a nucleus. In full-length insulin mole-
cules, there must be conformational changes for the LVEALYL
side chains of the segment to be exposed and to interact with
each other. Once nuclei are formed, full insulin molecules can
compete for sites on the nucleus with full insulin molecules and
can slow fibril growth.
Model for the Insulin Fibril, Based on the LVEALYL Crystal Structure.
Taken together, the x-ray fiber diffraction pattern of aligned
insulin fibrils and MPL measurements suggest important fea-
tures of fibrils of full-length insulin. The x-ray fiber diffraction
pattern reveals that the fibrils consist of ?-sheets running
parallel to the fibril axis with their ?-strands running perpen-
dicular (13). Thus, the 4.7 Å reflection in the x-ray diffraction
pattern (Fig. 6A) corresponds to the distance between the
strands in the ?-sheets. The two broad reflections centered at
11.7 Å and 9.0 Å on the equator (horizontal axis) correspond to
the distances between the sheets in the insulin fibril. These two
Model of the fibril
blue, respectively. The LVEALYL segment, which forms the spine of the fibril, is in dark blue. The SLYQLENY segment, from the A chain, which forms auxiliary
sheets to the spine of the fibril, is in dark red. Disulfide bonds are shown in yellow. (Middle) View down fibril axis of four ?-sheets of crystal structure of B chain
LVEALYL. The two sheets forming the dry steric zipper interface are in blue. Water molecules are shown as green spheres. (Right) View of fibril model, looking
down fibril axis. One layer of fibril model is made by stretching both monomers of native insulin (left) in a horizontal direction, converting the deep blue helix
of the B chain and the deep red helix of the A chain into extended ?-strands. These extended ?-strands are given the conformations of the four chain segments
of the crystal structure shown in the middle. Thus the spine of the fibril consists of a dry steric zipper formed by the mating of the central two LVEALYL strands
from the B chains of the two insulin molecules, plus two outer strands from the A chains of the two molecules.
Fibril model of insulin. (Left) Native structure of the insulin dimer (PDB code 1GUJ). A and B chains of insulin molecule are shown in pale red and pale
insulin fibrils. (A) Cross-? x-ray diffraction pattern of oriented insulin fibrils.
On the meridian (vertical axis), there is one strong reflection at 4.7 Å, corre-
on the equator (horizontal axis) are at 9.0 Å and 11.7 Å, arising from separa-
tions between ?-sheets. (B) Simulated fibril diffraction pattern calculated
from the model of the insulin fibril of Fig. 5. An excellent agreement of the
diffraction pattern of the model with the observed pattern is noticeable,
particularly the 4.7 Å reflection on the meridian and the 9.0 Å and 11.7 Å
reflections on the equator.
Comparison ofobserved and calculated fibril diffraction patterns of
Ivanova et al.PNAS ?
November 10, 2009 ?
vol. 106 ?
no. 45 ?
reflections might imply that there are two pairs of sheets with
different sheet-to-sheet distances, which form the fibrils. The
MPL measurements suggest that there are two insulin molecules
per 4.7 Å along the fibril (Fig. 4A).
Based on the findings above, the atomic structure of the B
chain segment LVEALYL was used as the molecular basis for
the steric zipper spine of the insulin fibril. Insulin fibrils are
primarily composed of ?-sheets (40), which implies that
LVEALYL undergoes a conversion from its native ?-helical
structure (shown in dark blue in Fig. 5, left panel) into an
extended ?-strand, as in the crystal structure of LVEALYL
(shown in dark blue in Fig. 5, middle panel). In our insulin fibril
model, two strands of LVEALYL from two insulin molecules
interdigitate to form one layer of the steric zipper spine of the
fibril. Thus, in the fibril of full-length insulin, LVEALYL
retains its conformation from the crystal structure (Fig. 5, right
The covalent constraints of the disulfide bonds linking the
B chain to the A chain (41) force the A chain also to convert
to an extended ?-strand upon extension of the LVEALYL
?-helix of the B chain (Fig. 5, right panel). The crystal packing
of LVEALYL molecules also provides a structural template
for the extended A chain segment LYQLENY. Thus, this A
chain segment adopts the backbone conformation of the
?-strands (colored in gray Fig. 5, middle panel) which lies
astride the ?-strands forming the dry steric zipper interface.
These two outer ?-sheets of the spine of insulin fibril are
formed by two stacks of LYQLENY segments of the A chain
(Fig. 5, right panel). The two B chain LVEALYL ?-sheets,
which form the dry steric zipper interface, lie between the two
sheets formed by LYQLENY. Because LYQLENY contains a
Tyr residue in the second position, this side chain superim-
poses on a Tyr from LVEALYL in the crystal structure,
preserving the ‘‘kissing tyrosine’’ interaction observed across
the wet interface of the crystal of LVEALYL (Fig. 5, middle
panel) in the fibril model of full-length insulin. These four
?-strands complete the model of the fibril spine.
The remaining segments of the A and B chains outside of the
fibril spine were modified from their native structure to fit within
the constraint of two molecules per 4.7 Å layer. Furthermore, to
be compatible with the 1,200 Å cross-over distance observed in
insulin fibrils, each layer of our model is given a left-hand twist
of ?0.71° with respect to the layer below. The result is the fibril
model shown in Fig. 5 (right panel).
In summary, the spine of our model contains four ?-sheets,
the inner pair forming a steric zipper that superimposes on our
crystal structure of LVEALYL from the B chain. The outer
two ?-sheets are formed from the segment LYQLENY of the
A chain, also found to contribute to the fibril spine. At the
periphery of the model, the N- and C-termini retain the
native-like structure of the insulin molecule. A similar model,
with a steric zipper spine and native-like structure on the
periphery, was proposed for a designed amyloid of ribonucle-
ase A (42).
A comparison of our model with the density of the 3D
cryo-EM reconstruction of insulin fibrils, determined by Jime-
nez et al. (18), is given in Fig. S2. The overall shape of the
electron density is captured well in most parts by our model
(Fig. S2, right). The parts of the model that do not fit the
density map are outside the spine of the fibril and possibly
disordered, which could explain why they are not observed in
the low-resolution density map. Moreover, the calculated MPL
of insulin fibrils, including only the residues from the core of
the fibrils, is within experimental error of the value obtained
by Jimenez et al. (18) (for more details, see SI Text). Thus,
we can conclude that our model agrees with the cryo EM
Assessment of Our Insulin Fibril Model. The model has been built to
be consistent with several structural and biochemical observa-
tions. First, its structure with two insulin molecules per 4.7 Å
layer of the fibril is consistent with our MPL measurements (Fig.
4). It was also found that three dimers of insulin comprise the
fibril precursors (43). Thus the fibril growth proceeds via
stacking of these precursors along the fibril axis, suggesting that
insulin fibrils are formed of dimers repeating along the length of
the fibrils in agreement with our MPL measurements. Second,
the simulated fiber diffraction pattern (Fig. 6B) computed from
our model corresponds well to the observed cross-? pattern of
oriented insulin fibrils (Fig. 6A). Of note, two equatorial reflec-
tions, which arise from sheet-to-sheet interactions, are present in
both the simulated and observed diffraction patterns at 11.7 Å
and 9.0 Å. (Fig. 6). Third, our previous finding that crystals of
that the crystal structure of the peptide resembles the structural
organization of the fibril spine. This is the basis for our use of the
structure of LVEALYL as a template for the spine of the
full-length insulin fibrils. Fourth, the model is consistent with
previous findings (4, 17, 20, 21) and our observation that the B
chain is important for fibril formation: the B chain forms fibrils
independently and can also delay and accelerate fibril forma-
tion. Fifth, our model incorporates the LYQLENY segment of
the A chain on the periphery of the spine. This is consistent
with finding that segments from the A chain (residues A13–
A19) and B chain (residues B9–B19) are protected against
proton exchange (25). Sixth, inclusion of the Glu residues of
LVEALYL and LYQLEN in the spine of our model may
explain the more rapid fibrillation of insulin at pH 2.5, where
the Glu is expected to be largely uncharged, than at neutral pH,
where it carries a negative charge. Thus our model for the
insulin fibril is supported by much structural and biochemical
Materials and Methods
Polymerization Assays. Insulin was purchased from Sigma-Aldrich. Peptides
assays were performed with 0.25 mM insulin in 50-mM glycine buffer pH 2.5.
reaction was monitored by ThioflavinT (ThT) fluorescence (for more informa-
tion, see the SI Text).
Electron Microscopy. For details on EM, see the SI Text.
Crystallization of LVEALVL. Crystals of LVEALYL grew in a hanging drop after
mixing 2 ?l of 1.82-mM peptide with 1 ?l crystallization solution containing
20% MPD/0.1 M sodium citrate pH 5.5 at room temperature.
X-Ray Crystallographic Data Collection and Processing. X-ray diffraction data
sets were collected at the Swiss Light Source beamline X10SA, equipped with
a MAR CCD detector. Data were collected in 5° wedges at a wavelength of
0.97645 Å using a 5 ?m beam diameter. For more details see Table S2 and SI
Preparation of Oriented Samples and X-Ray Diffraction. For details on prepa-
ration of oriented samples and x-ray diffraction, see the SI Text
Model Construction. The model of the insulin fibril was built directly from the
crystal structure of the segment LVEALYL (insulin B chain residues 11–17).
Model building was performed with the graphics program O (44). This crude
model was energy minimized using the program CNS (45) with van der Waals,
electrostatic, and hydrogen bonding terms (46) (for more details, see the SI
Simulation of Fibril Diffraction. Simulated fibril patterns of fibril models were
produced by cylindrical averaging of the single crystal diffraction intensities
(for more information, refer to the SI Text).
www.pnas.org?cgi?doi?10.1073?pnas.0910080106Ivanova et al.
STEM Sample Preparation and Data Processing. The samples were prepared at
the STEM facility at the Brookhaven National Laboratory (for more informa-
tion, see the SI Text and Figs. S3 and S4).
ACKNOWLEDGMENTS. We thank Drs. Andrew D. Miranker, Lukasz Salwin-
ski, Ruben Diaz-Avalos, and Ivaylo Dinov for discussion; Dr. Martha Simon
and Beth Lin of Brookhaven National Laboratory for making STEM mea-
surements and preparing specimens; Dr. Helen Saibil for providing a
cryo-EM reconstruction of insulin fibrils; Prof. Ehmke Pohl and the staff at
the staff at the Advanced Photon Source beamline 24-ID-E. We thank the
National Science Foundation, the Department of Energy/Office of Biolog-
ical and Environmental Research, the National Institutes of Health, and
Howard Hughes Medical Institute for support. S.A.S. was supported by a
University of California Los Angeles National Science Foundation Integra-
tive Graduate Education and Research Traineeship. BNL STEM is supported
by Department of Energy/Office of Health and Environment Research.
1. Westermark P, et al. (2005) Amyloid: Toward terminology clarification. Report from
the Nomenclature Committee of the International Society of Amyloidosis. Amyloid
2. Dische FE, et al. (1988) Insulin as an amyloid-fibril protein at sites of repeated insulin
injections in a diabetic patient. Diabetologia 31:158–161.
3. Storkel S, Schneider HM, Muntefering H, Kashiwagi S (1983) Iatrogenic, insulin-
dependent, local amyloidosis. Lab Invest 48:108–111.
4. Brange J, Andersen L, Laursen ED, Meyn G, Rasmussen E (1997) Toward understanding
insulin fibrillation. J Pharm Sci 86:517–525.
5. Westermark P (2005) Aspects on human amyloid forms and their fibril polypeptides.
FEBS J 272:5942–5949.
6. Astbury WT, Dickinson S (1935) The x-ray interpretation of denaturation and the
structure of the seed globulins. Biochem J 29:2351–2360.
7. Geddes AJ, Parker KD, Atkins ED, Beighton E (1968) ‘‘Cross-beta’’ conformation in
proteins. J Mol Biol 32:343–358.
8. Sunde M, Blake C (1997) The structure of amyloid fibrils by electron microscopy and
x-ray diffraction. Adv Protein Chem 50:123–159.
9. Wilhelm KR, et al. (2007) Immune reactivity towards insulin, its amyloid and protein
S100B in blood sera of Parkinson’s disease patients. Eur J Neurol 14:327–334.
10. Ahmad A, Uversky VN, Hong D, Fink AL (2005) Early events in the fibrillation of
monomeric insulin. J Biol Chem 280:42669–42675.
11. Bouchard M, Zurdo J, Nettleton EJ, Dobson CM, Robinson CV (2000) Formation of
insulin amyloid fibrils followed by FTIR simultaneously with CD and electron micros-
copy. Protein Sci 9:1960–1967.
12. Nettleton EJ, et al. (2000) Characterization of the oligomeric states of insulin in
self-assembly and amyloid fibril formation by mass spectrometry. Biophys J 79:1053–
13. Burke MJ, Rougvie MA (1972) Cross-beta protein structures. I. Insulin fibrils. Biochem-
Structures of insulin fibrils, glucagon fibrils, and intact calf lens. Arch Biochem Biophys
15. Turnell WG, Finch JT (1992) Binding of the dye congo red to the amyloid protein pig
insulin reveals a novel homology amongst amyloid-forming peptide sequences. J Mol
16. Sawaya MR, et al. (2007) Atomic structures of amyloid cross-beta spines reveal varied
steric zippers. Nature 447:453–457.
17. Brange J, Dodson GG, Edwards DJ, Holden PH, Whittingham JL (1997) A model of
insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despen-
tapeptide insulin). Proteins 27:507–516.
Acad Sci USA 99:9196–9201.
19. Vestergaard B, et al. (2007) A helical structural nucleus is the primary elongating unit
of insulin amyloid fibrils. PLoS Biol 5:e134.
20. Gibson TJ, Murphy RM (2006) Inhibition of insulin fibrillogenesis with targeted pep-
tides. Protein Sci 15:1133–1141.
21. Nielsen L, Frokjaer S, Brange J, Uversky VN, Fink AL (2001) Probing the mechanism of
insulin fibril formation with insulin mutants. Biochemistry 40:8397–8409.
22. Devlin GL, et al. (2006) The component polypeptide chains of bovine insulin nucleate
or inhibit aggregation of the parent protein in a conformation-dependent manner. J
Mol Biol 360:497–509.
23. Hong DP, Fink AL (2005) Independent heterologous fibrillation of insulin and its
B-chain peptide. Biochemistry 44:16701–16709.
24. Ivanova MI, Thompson MJ, Eisenberg D (2006) A systematic screen of beta(2)-
25. Tito P, Nettleton EJ, Robinson CV (2000) Dissecting the hydrogen exchange properties
spectrometry. J Mol Biol 303:267–278.
prion Sup35 reveals a dehydrated beta-sheet structure for amyloid. Proc Natl Acad Sci
Proc Natl Acad Sci USA 101:87–92.
28. Tenidis K, et al. (2000) Identification of a penta- and hexapeptide of islet amyloid
polypeptide (IAPP) with amyloidogenic and cytotoxic properties. J Mol Biol 295:1055–
29. Reches M, Porat Y, Gazit E (2002) Amyloid fibril formation by pentapeptide and
tetrapeptide fragments of human calcitonin. J Biol Chem 277:35475–35480.
propensity are necessary for amyloid fibril formation from tetrapeptides. J Biol Chem
31. Tjernberg LO, et al. (1996) Arrest of beta-amyloid fibril formation by a pentapeptide
ligand. J Biol Chem 271:8545–8548.
32. Findeis MA, et al. (1999) Modified-peptide inhibitors of amyloid beta-peptide poly-
merization. Biochemistry 38:6791–6800.
33. Gilead S, Gazit E (2004) Inhibition of amyloid fibril formation by peptide analogues
modified with alpha-aminoisobutyric acid. Angew Chem Int Ed Engl 43:4041–4044.
34. Kapurniotu A, Schmauder A, Tenidis K (2002) Structure-based design and study of
IAPP amyloid formation and cytotoxicity. J Mol Biol 315:339–350.
35. Chabry J, Caughey B, Chesebro B (1998) Specific inhibition of in vitro formation of
protease-resistant prion protein by synthetic peptides. J Biol Chem 273:13203–13207.
36. Ma J, Yee A, Brewer HB, Jr, Das S, Potter H (1994) Amyloid-associated proteins alpha
1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-
protein into filaments. Nature 372:92–94.
37. Eriksson S, Janciauskiene S, Lannfelt L (1995) Alpha 1-antichymotrypsin regulates
Alzheimer beta-amyloid peptide fibril formation. Proc Natl Acad Sci USA 92:2313–
38. Evans KC, Berger EP, Cho CG, Weisgraber KH, Lansbury PT, Jr (1995) Apolipoprotein E
is a kinetic but not a thermodynamic inhibitor of amyloid formation: Implications for
the pathogenesis and treatment of Alzheimer disease. Proc Natl Acad Sci USA 92:763–
39. Wisniewski T, Castano EM, Golabek A, Vogel T, Frangione B (1994) Acceleration of
Alzheimer’s fibril formation by apolipoprotein E in vitro. Am J Pathol 145:1030–1035.
40. Nielsen L, Frokjaer S, Carpenter JF, Brange J (2001) Studies of the structure of insulin
fibrils by Fourier transform infrared (FTIR) spectroscopy and electron microscopy.
J Pharm Sci 90:29–37.
41. Nettleton EJ (1998) Ph.D. thesis. (Oxford University, Oxford).
42. Sambashivan S, Liu Y, Sawaya MR, Gingery M, Eisenberg D (2005) Amyloid-like fibrils
of ribonuclease A with three-dimensional domain-swapped and native-like structure.
43. Nayak A, Sorci M, Krueger S, Belfort G (2009) A universal pathway for amyloid nucleus
and precursor formation for insulin. Proteins 74:556–565.
44. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building
protein models in electron density maps and the location of errors in these models.
Acta Crystallogr A 47:110–119.
45. Brunger AT, et al. (1998) Crystallography & NMR system: A new software suite for
macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–
46. Fabiola F, Bertram R, Korostelev A, Chapman MS (2002) An improved hydrogen bond
potential: Impact on medium resolution protein structures. Protein Sci 11:1415–1423.
Ivanova et al.PNAS ?
November 10, 2009 ?
vol. 106 ?
no. 45 ?