Stable and metastable states of human amylin in solution.

Abstract

Patients with type II diabetes exhibit fibrillar deposits of human amylin protein in the pancreas. It has been proposed that amylin oligomers arising along the aggregation or fibril-formation pathways are important in the genesis of the disease. In a step toward understanding these aggregation pathways, in this work we report the conformational preferences of human amylin monomer in solution using molecular simulations and infrared experiments. In particular, we identify a stable conformer that could play a key role in aggregation. We find that amylin adopts three stable conformations: one with an α-helical segment comprising residues 9-17 and a short antiparallel β-sheet comprising residues 24-28 and 31-35; one with an extended antiparallel β-hairpin with the turn region comprising residues 20-23; and one with no particular structure. Using detailed calculations, we determine the relative stability of these various conformations, finding that the β-hairpin conformation is the most stable, followed by the α-helical conformation, and then the unstructured coil. To test our predicted structure, we calculate its infrared spectrum in the amide I stretch regime, which is sensitive to secondary structure through vibrational couplings and linewidths, and compare it to experiment. We find that theoretically predicted spectra are in good agreement with the experimental line shapes presented herein. The implications of the monomer secondary structures on its aggregation pathway and on its interaction with cell membranes are discussed.

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Stable and Metastable States of Human Amylin in Solution
Allam S. Reddy,†Lu Wang,‡Sadanand Singh,†Yun L. Ling,‡Lauren Buchanan,‡Martin T. Zanni,‡
James L. Skinner,‡and Juan J. de Pablo†*
Departments of†Chemical and Biological Engineering and‡Chemistry, University of Wisconsin-Madison, Madison, Wisconsin
ABSTRACT
proposed that amylin oligomers arising along the aggregation or fibril-formation pathways are important in the genesis of the
disease. In a step toward understanding these aggregation pathways, in this work we report the conformational preferences
of human amylin monomer in solution using molecular simulations and infrared experiments. In particular, we identify a stable
conformer that could play a key role in aggregation. We find that amylin adopts three stable conformations: one with an a-helical
segment comprising residues 9–17 and a short antiparallel b-sheet comprising residues 24–28 and 31–35; one with an extended
antiparallel b-hairpin with the turn region comprising residues 20–23; and one with no particular structure. Using detailed calcu-
lations, we determine the relative stability of these various conformations, finding that the b-hairpin conformation is the most
stable, followed by the a-helical conformation, and then the unstructured coil. To test our predicted structure, we calculate its
infrared spectrum in the amide I stretch regime, which is sensitive to secondary structure through vibrational couplings and line-
widths, and compare it to experiment. We find that theoretically predicted spectra are in good agreement with the experimental
line shapes presented herein. The implications of the monomer secondary structures on its aggregation pathway and on its inter-
action with cell membranes are discussed.
Patients with type II diabetes exhibit fibrillar deposits of human amylin protein in the pancreas. It has been
INTRODUCTION
A broad range of human diseases including Huntington’s,
Alzheimer’s, Creutzfeldt -Jacob’s, and type II diabetes are
associated with misfolding and aggregation of a specific
protein or proteins (1,2). In these diseases, the proteins in
question are known to convert from their native functional
state into highly organized fibrillar aggregates. Recent
investigations have shown that intermediate states that occur
along the formation pathway of fibrillar aggregates induce
apoptosis of their associated cells, leading to disease
symptoms (3–6). Hence, a deeper understanding of the
aggregation process would provide useful information for
development of therapeutic strategies that could inhibit
aggregation and maybe even reverse it.
Its importance notwithstanding, the structure of most of
the proteins involved in protein aggregation diseases is not
known with atomic-level resolution. This is partly because
most of these proteins are membrane-bound and exhibit
extremely low solubility. This makes their structural charac-
terizationbytraditionalexperimentalmeans,includingx-ray
crystallography and nuclear magnetic resonance (NMR),
particularly demanding. Furthermore, these proteins exhibit
very fast aggregation kinetics, thereby complicating the
interpretation of experimental data considerably.
In the particular case of type II diabetes, the culprit
is amylin, also called human islet amyloid polypeptide
(hIAPP), a 37-residue hormone produced by the islet b-cells
in the pancreas whose sequence is shown in references 7–9.
The aggregation of amylin is widely perceived to be associ-
ated with many of the disease’s symptoms (10,11). Past
efforts have been largely aimed at characterizing the fibrillar
aggregates formed by this protein. Using x-ray and electron
diffraction techniques, Sumner-Makin and Serpell (12)
determined that the fibrils of the human amylin protein are
made up of extended b-strands that run perpendicular to
the fibril axis. Jayasinghe and Langen (13), using electron
paramagnetic resonance spectroscopy, determined that the
b-strands adopt a parallel orientation. Similar results were
also obtained using two-dimensional infrared measurements
(14,15). More recently, using solid-state NMR, it has been
proposed that amylin aggregates form a symmetrical struc-
ture consisting of striated ribbons containing layers of
parallel b-sheet (16). The structure of amylin aggregates
has also been studied using molecular models (17,18) and,
consistent with experimental observations, past simulations
have identified b-sheet structures with interdigitated side
chains.
Although some consensus is starting to emerge regarding
the structure of the human amylin aggregates, less is known
about the structure of the individual hIAPP molecule in
solution and the early-stage aggregates that are thought to
precede fibril formation. Recent experiments have produced
evidence for the occurrence of a-helical intermediates of
amylin in the presence of membranes and for associations
of oligomers in the lag phase of aggregation (19–22). As
shown in this work, our calculations predict that roughly
one-third of the ensemble of structures that arises in dilute
solution consists of conformations with a partial a-helix,
which may be responsible for these experimental observa-
tions, or it could be that a structure change occurs upon
interactions with membranes or other peptides.
Submitted April 14, 2010, and accepted for publication July 6, 2010.
*Correspondence: depablo@engr.wisc.edu
Editor: Gregory A. Voth.
? 2010 by the Biophysical Society
0006-3495/10/10/2208/9$2.00
doi: 10.1016/j.bpj.2010.07.014
2208Biophysical Journal Volume 99October 20102208–2216

Page 2

Our approach is to generate an atomic level model of the
amylin monomer using advanced atomistic molecular simu-
lations in explicit water, which can then be tested against
experimentbycalculatinginfraredspectrafromtheresulting
molecular structures. Infrared spectra by themselves cannot
provide atomic level structures, but because the backbone
vibrational modes are strongly coupled to one another and
because the linewidths are sensitive to solvent exposure
(14,15,23,24), the infrared (IR) spectra reflect the secondary
structure content of proteins and polypeptides. For instance,
b-sheetpolypeptidesabsorbnear1620cm–1andhavenarrow
linewidths,whereasrandomcoilsabsorbnear1645cm–1and
are broad. Peak fitting is often used to extract secondary
structure content from IR spectra, but the accuracy of the
conclusions is often suspect because the fits are usually not
unique and because the peaks have no rigorous theoretical
basis. However, IR spectra can be quantitatively predicted
from structures generated by molecular simulations through
semiclassicallineshapetheoriesandbyincludingvibrational
couplings,which istheapproachwe usehere.Thiscombina-
tion of simulation, theory, and experiment offers advantages
over more established techniques. For instance, the interpre-
tation of circular dichroism spectra, which is perhaps the
most often used spectroscopy for amyloids, is entirely
empirical. And NMR and x-ray spectroscopy continue to
be difficult to apply to amyloids, particularly in situations
where misfolding and aggregation occur over timescales
that are faster than the time required for experimental
measurements. Our approach is thus unique in that it enables
a quantitativecycle of structure prediction and validation for
challenging systems such as amyloid aggregation.
Theoretical secondary structure prediction algorithms
predict that, in solution, the human amylin polypeptide
adopts an a-helical secondary structure that spans residues
8–14 (25). However, invitro circular dichroism experiments
suggest that human amylin is natively unstructured (26–30).
Experimentally it is also known that when human amylin is
dissolved in hexafluoro-isopropanol (HFIP), it is found in
a predominantly monomeric state with a stable a-helical
domain spanning residues 5–20 (31). Similarly, in solution,
the calcitonin-gene-related peptide, which has a sequence
similar to that of human amylin and belongs to the same
hormone family, forms a stable a-helical segment spanning
residues 8–18 (32). Recently, based on NMR experiments
and by using better aggregate removal protocols, Yonemoto
et al. (33) and Cort et al. (34) showed that human amylin has
a strong preference for an a-helical secondary structure near
the N-terminal end of the peptide. In their study, to mini-
mize aggregation the authors use a low temperature of
5?Cand pHof 6. Note that other experiments inthe presence
of membranes also seem to support this secondary structure
for the human amylin protein (20,36–38). Very recently,
Dupuis et al. (42) studied the hIAPP monomer using a com-
bination of ion mobility mass spectrometry and all-atom
replica exchange molecular dynamics simulations with
implicit water and found three distinct confomational fami-
lies, one of which is an extended b-hairpin and one of which
is a compact helix-coil.
When studying hIAPP, it is useful to consider the partic-
ular sequence of the amylin peptide that arises in rats, called
rIAPP. The rat amylin peptide exhibits >80% sequence
homology with hIAPP (7,8), and yet its propensity to form
aggregates is severely diminished, thereby facilitating
studies of the monomer in solution (29). Experiments
suggest that rat and human amylin proteins interact simi-
larly with lipid monolayers (39), and it is therefore thought
that the human and rat proteins may have several common
structural features. High-resolution NMR studies show
that rIAPP adopts an a-helical conformation spanning resi-
dues 5–19 (40). In the presence of phospholipid micelles,
the N-terminus adopts a helical structure (41). Based on
available evidence for the rat amylin protein, one could
expect the human variant to also exhibit an a-helical struc-
turenearitsN-terminus.Inrecentwork,usingacombination
of replica exchange umbrella sampling simulations and IR
spectroscopy, we have shown that the folded state of the
rat amylin protein exhibits an a-helical secondary structure
between residues 7–17 (8). That work also served to estab-
lish the accuracy of our molecular simulation advanced
sampling algorithms and our protocol for converting the
results of simulations into IR spectra; we predicted NMR
chemical shifts and IR spectra, and both were found to be
in good agreement with experiment. Our results on the
structure of rIAPP confirm the recent results of Dupuis
et al. (42).
In this work, using all-atom replica exchange molecular
dynamics with explicit water, we determine the stable conf-
ormations of hIAPP in solution and provide quantitative
predictions of their relative stability.
We also compare our findings for human amylin to results
for rat amylin, and propose an explanation for the proclivity
of human amylin to form aggregates in solution. We do so
by resorting to what we believe is a novel simulation tech-
nique, developed for this work, that enables extensive
sampling and calculation of free energies for different
conformers of amylin. We then employ the line shape theory
to predict the vibrational absorption spectrum, and compare
it to our own Fourier-transform infrared (FTIR) measure-
ments. Our results indicate that the human peptide can adopt
a-helical, random coil, and b-hairpin conformations in solu-
tion. The relative abundance of these conformers at room
temperature is 31%, 29%, and 40%, respectively. The simu-
lated and measured spectra are in quantitative agreement. In
contrast, in our previous work we found that the rat peptide
can only adopt a-helical and random coil conformations
(with relative abundances of 55% and 45%, respectively)
(8). Based on these findings, we propose that the ability of
human amylin to adopt a stable b-hairpin is a crucial char-
acteristic of the molecule that facilitates its ability to form
fibrils, and that such a conformer is a key intermediate.
Biophysical Journal 99(7) 2208–2216
Structure of Human Amylin Peptide2209

Page 3

MATERIALS AND METHODS
Molecular simulations
The amino-acid sequence of the human amylin protein studied in our work
is given in Green et al. (7) and Reddy et al. (8). Consistent with experi-
ments, the C-terminal of the peptide is capped with an NH2group, and a
disulfide bond is present between CYS-2 and CYS-7 residues. The protein
is modeled using the GROMOS96 53a6 force field, while the simple point-
charge water model is adopted for water (43). The ionization state of the
amino-acid side chains was assigned based on their pKavalues. The result-
ing peptide structure was found to have a net positive charge of þ3. To
make the system charge neutral, three Cl?ions were added to the simula-
tion box. The GROMOS96 53a6 force field has been used extensively in
the literature to study the folding of protein molecules, and was found to
provide good agreement with experiment in our recent studies of peptide
aggregation and of rIAPP (8).
Molecular simulations were performed with the GROMACS molecular
simulation package (44,45). Long-range electrostatic interactions were
treated with a particle-mesh Ewald sum (46,47). All simulations were
performed with rigid bonds (using the linear constraint solver method)
and with an integration time step of 2 fs. Once at equilibrium, most simu-
lations were performed at a temperature of 298 K and pressure of 1 bar
using Berendsen coupling (48).
Replica-exchange method
The replica exchange molecular dynamics (REMD) or parallel tempering
method has been used extensively in the literature to understand the confor-
mational preferences of proteins (49–51). The basic idea of REMD is to
simulate copies (replicas) of the same system at different temperatures
values. The configurations from adjacent temperature replicas are
exchanged at regular intervals using the Metropolis acceptance criteria
(Pacc) given below:
Pacc ¼ minð1;expðDbDEÞÞ:
(1)
In the above equation, Db and DE represent the difference in the inverse
temperature, and the difference in total internal energy of the replicas being
considered for an exchange of configurations, respectively.
We used a total of 74 replicas spanning a temperature range of 273–
578 K. The simulation length for each replica was 10 ns, with a total simu-
lation length of 740 ns. The exchange moves between adjacent replicas
were attempted every 2 ps. The optimal performance of the algorithm
requires that the probability distribution curves of the potential energies
corresponding to neighboring replicas exhibit a sufficient degree of overlap.
Fig. S1 in the SupportingMaterial shows the potential energy distribution at
the different temperatures used in our REMD simulations. As can be seen
from the figure, the overlap of the potential energy between adjacent
replicas was chosen such that it leads to an average acceptance rate of
20% (51).
Stable conformations of the peptide were extracted from our REMD
simulations by resorting to principal component analysis (52–54). As
discussed below, our analysis revealed the presence of three major clusters
corresponding to the three conformations discussed in this article. Fig. S2
provides the results of that analysis.
Thermodynamic integration
Our REMD simulations indicate the human peptide can adopt a-helical,
random coil, and b-hairpin conformations in solution (see Results).
The relative free energy (and thermodynamic stability) of these states
was determined using thermodynamic integration (TI). The TI scheme
used here is similar to that used in the context of simulations of polymeric
materials to identify order-disorder transitions (55). For the human peptide,
this technique is used to determine the free energy difference between 1), an
a-helical conformation and a random coil, and 2), a b-hairpin conformation
and a random coil.
In our previous we work we have shown that the rat amylin protein exists
in solution in the a-helicaland random coil conformations. Forcomparison,
we also used TI to calculate the free energy difference between an a-helical
and a random coil conformation of the rat amylin peptide.
The TI pathway used in our problem is illustrated in Fig. S3. As shown in
the figure, it consists of four segments. Starting from the folded state, along
the segment labeled 1, an external umbrella potential Ufis imposed whose
relative strength (l) is linearly increased from zero to unity, as one travels
from vertex a to vertex b. Along the segment labeled 2, denoted by a line
connecting vertices b and c, the temperature of the system was gradually
raised from 298 K to 498 K. Next, along Segment 3, denoted by the line
connecting c and d, the relative strength (l) of the imposed umbrella
potential, Uf, was gradually reduced from unity to zero. Finally, along
Segment 4, the temperature of the system was gradually lowered from
498 K back to 298 K.
In the particular case of islet amyloid polypeptide, we found it sufficient
to use an umbrella potential, Uf, consisting of a set of harmonic springs
imposed between the Caatoms of the native contacts. The functional
form of Ufis simply
Uf ¼ lk
X
N
j¼1
?rj? r0
?2;
(2)
where the spring constant was chosen to be equal to k ¼ 500 kJ/mol/nm2.
The symbol r0represents the equilibrium distance between the contacts for
an ideally folded structure, the symbol rjrepresents the actual distance
between the Caatoms of the jthcontact, and N represents the total number
of contacts. For an a-helical conformation, a native contact is defined as
that between two residues which are three residues apart (i and i þ 4).
The distance r0that corresponds to an ideal helix was set 0.59 nm. In the
case of a b-hairpin, a native contact was defined as that between the two
residues that are involved in hydrogen bonding in the b-sheet. The corre-
sponding r0for b-sheet structure was set to 0.55 nm (based on an ideal
b-sheet). Using these criteria, and based on results from exploratory equi-
librium MD simulations (see Results), for an a-helical conformation a total
of N ¼ 7 umbrellas were used between residue pairs (7,11), (8,12), (9,13),
(10,14), (11,15), (12,16), and (13,17). Similarly, for the b-hairpin structure,
N ¼ 7 umbrellas were used between residue pairs (13,28), (14,27), (15,26),
(16,25), (17,24), (18,23), and (19,24).
IR line shape calculations
We followed the protocol used for rat amylin (8,56) to determine the IR
amide I line shape of human amylin. However, herein we use an improved
electrostatic amide I backbone map, and a new, to our knowledge, side-
chain map for glutamine and asparagine residues, which were developed
from experimental spectra of n-methyl-acetamide and acetamide, respec-
tively, in various solvents, and the total simulation time was 10 ns. For
backbone chromophores,
ui; b ¼ uiþ Dui; NðFi?1; i;Ji?1; iÞ
þ Dui; CðFi; iþ1;Ji; iþ1Þ;
(3)
with
ui ¼ 1684 þ 7729Ei; C? 3576Ei; N:
(4)
In Eq. 3, Ei, Cis the electric-field component for the ithchromophore in the
C¼O direction on the C atom and Ei, Nis that on the N atom (both in atomic
units), and the frequency is in cm–1. The exclusion list for the electric field
calculation includes backbone atoms in the (i-1)th, ith, and (iþ1)thpeptide
Biophysical Journal 99(7) 2208–2216
2210Reddy et al.

Page 4

units. To account for contributions from the nearest-neighbor peptide units,
we have adapted the nearest-neighbor frequency shift map developed by la
Cour Jansen et al. (57). These contributions depend on the (F, J) angles
between peptide units and are denoted as Dui, Nand Dui, C. The side-chain
frequencies are calculated as
ui; s ¼ 1714 þ 2154Ei; Cþ 3071Ei; N:
Couplings between adjacent amide groups have been determined by the
nearest-neighbor coupling map of la Cour Jansen et al. (57). All the other
coupling constants have been calculated from the transition dipole coupling
method (58–61), and we have adopted the parameters optimized by Torii
and Tasumi (62).
(5)
Experimental peptide preparation
and IR spectrum
Peptide preparation
hIAPP were purchased from Bachem (Bubendorf, Switzerland) and dis-
solved in deuterated hexafluoroisopropanol (d-HFIP) at a concentration
of 0.5 mM. An aliquot of the d-HFIP stock solution was made into D2O
solution after evaporating d-HFIP. The hIAPP D2O solution was dialyzed
in 0.1 mM DCl multiple times and then one time in D2O to remove residual
TFA. The sample was then lyophilized and redissolved in d-HFIP. The
denaturant HFIP was then removed by evaporation under a stream of
nitrogen to generate a film in Eppendorf tubes. Amyloid formation was
initiated by redissolving the film in D2O at pH 6. The initial peptide
concentration was 0.25 mM.
Fourier-transform infrared
Infrared absorption data were taken by a Mattson Instruments (Thermo
Electron, Waltham, MA) Galaxy series Fourier-transform infrared (FTIR)
7000 spectrometer purged with dry air. The sample was placed between
two CaF2plates separated by a 100-mM Teflon spacer. The spectrum was
collected immediately after redissolving the film in D2O at pH 6, as
mentioned above in Peptide Preparation. It was smoothed by truncating
higher frequency noises in time domain.
RESULTS AND DISCUSSION
A principal component analysis performed on results from
extensive REMD simulations revealed three stable confor-
mations for the peptide:
1. An a-helical conformation where residues 9–17 were
folded into an a-helix and a short b-sheet between
residues 24–28 and 31–35 (Fig. 1 a).
2. A b-hairpin conformation with a turn at residues between
20 and 23 (Fig. 1 b).
3. A random coil conformation (Fig. 1 c).
The a-helical conformation was found to be similar to that
predicted for the rat amylin protein (8) (Fig. 1 d). Our three
stable hIAPP conformations are very similar to those found
by Dupuis et al. (42). The b-sheet domain in the a-helical
conformation includes the region with proline mutations in
rat amylin protein. The low amyloidogenicity associated
with the proline residues might explain the difference in
the structure of human amylin protein from rat amylin
protein. Calcitonin-gene-related peptide is known to exist
in an a-helical conformation in its active form, and loses its
activity upon the loss of a-helicity (63).
Long molecular dynamics simulations (100 ns) of human
amylin revealed that the a-helical conformation can sponta-
neously convert into the b-hairpin conformation (observed
ab
cd
FIGURE 1
(a) a-helical conformation, (b) b-hairpin conformation, (c) random coil
conformation, and (d) rat amylin in a-helical conformation (8).
Representative configurations of human amylin peptide in
Biophysical Journal 99(7) 2208–2216
Structure of Human Amylin Peptide2211

Page 5

in two out of four simulations). In contrast, MD simulations
started from the b-hairpin conformation were always found
to be stable (average RMSD < 3 A˚). Several snapshots of
the secondary structure of the molecule at different times,
along one of the misfolding pathways, are presented in
Fig. 2 a. The b-hairpin order parameter, measured as the
fraction of native b-hairpin contacts along the misfolding
pathway, is presented in Fig. 2 b. As can be seen in Fig. 2
a, the early steps in the misfolding pathway of hIAPP
involve loss of contacts between residues 24–28 and 31–
35, which in the folded state of the protein correspond to
a short b-sheet. This event is followed by residues 24–28
forming new contacts with residues 15–19, leading to the
formation of the b-hairpin structure.
Having identified candidate structures for the a-helical
and b-hairpin states of hIAPP in solution, we proceeded to
investigate their relative stability as a function of suitable
order parameters (see Materials and Methods) by calcu-
lating their free energy vis-a `-vis that of the random coil
state. To that end, we performed extensive TI simulations
using a newly proposed algorithm. Table 1 shows the free
energy changes of the protein during the a-helix-coil and
b-hairpin-coil transformations. Our results show that an
a-helix is preferred over a random coil structure. However,
our results also show that the b-hairpin is more favorable
than both the random coil (?0.77 kJ/mol) and the a-helix
(?0.6 kJ/mol). Using this method, we also calculated the
free energy during the a-helix-coil transformation of rat
amylin protein (?0.47 kJ/mol). This value is consistent
with our previously published value, where calculations
were performed using a very different simulation technique
(8). In Table 2, we report the enthalpy change (DH)
associated with the transitions studied in the TI. The DH
is computed as the difference between the average potential
energy of the two end states in TI. Our results show that
the a-helical and b-hairpin conformations are stabilized
enthalpically, and preferred over the random coil conforma-
tions. The enthalpy gain for the a-helical conformation
is greater than that for the b-hairpin. However, there is an
entropic cost associated with folding the protein from
a random coil into the a-helical and b-hairpin confor-
mations. These results indicate that the a-helical conforma-
tion, which is stabilized by enthalpic contributions to the
free energy, would be even more stable and more pro-
nounced at lower temperatures. This observation is consis-
tent with the recent NMR studies by Yonemoto et al. (33),
where a larger a-helical content was observed for the
peptide at 5?C.
In Table 2, we also report average number of protein-
protein and protein-water hydrogen bonds. A hydrogen
bond is defined based on a acceptor-donor-hydrogen cutoff
angle of 30?and a cutoff distance of 0.35 nm between
hydrogen-acceptors (64,65). Our results indicate that the
a-helical and b-hairpin states are primarily stabilized by in-
trapeptide hydrogen bonds. The random coil state on the
other hand, is stabilized by peptide-water hydrogen bonds.
The calculated IR spectrum for each conformer (see
Materials and Methods) is shown in Fig. 3. The spectra
for the a-helical and b-hairpin states peak at ~1644 and
1643 cm–1, respectively, whereas the spectrum for the
random coil peaks at ~1657 cm–1. We calculated the
FIGURE 2
along a representative folding trajectory at (i) 0 ns, (ii) 50 ns, and (iii)
80 ns. (b) b-Hairpin order parameter (measured as fraction of native
contacts in b-hairpin) along the representative trajectory.
(a) Representative configurations of human amylin peptide
TABLE 1
and human amylin proteins calculated using TI, as explained in
the text
Free energy changes during folding of rat amylin
ProteinTransitionFree energy (kJ/mol)
Rat amylin
Human amylin
Human amylin
DF (random coil / a-helix)
DF (random coil / a-helix)
DF (random coil / b-hairpin)
?0.47 5 0.08
?0.17 5 0.09
?0.77 5 0.08
TABLE 2
changes for human amylin protein
Enthalpy, entropy, and number of hydrogen-bond
Transition
DH (kJ/mol) TDS (kJ/mol) DNHB
protein?proteinDNHB
5.669
protein?water
?11.907Random coil
/ a-helix
Random coil
/ b-hairpin
?78.3
?78.13
?70.1
?69.33 4.664
?11.350
For random coil conformation, DNHB
101:32.
protein?protein¼ 12.032, DNHB
protein?water¼
Biophysical Journal 99(7) 2208–2216
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weighted average of these spectra using the relative popula-
tions (listed earlier) that follow from the relative free
energies determined above. As shown in Fig. 3, the theoret-
ical line shape is in quantitative agreement with experiment
(see Materials and Methods), consistent with our contention
that there is a substantial population of the b-hairpin
conformer in solution. Note that our experimental line shape
showed only minor changes when the amylin concentration
was doubled, and that a similar experimental line shape
for very dilute (0.01 mM) amylin solution at early times
after mixing has been determined by Jha et al. (30), thus
providing some confirming evidence that our spectrum is
for the amylin monomer. Also, note that the differences in
the IR spectra of the three conformations are caused by
the vibrational couplings between residues. Standard
interpretation of IR spectra based on empirical rules would
not assign a peak at 1643 cm–1to a b-sheet, because
b-sheets usually absorb at 1620 cm–1. The b-hairpin absorbs
much higher, because the vibrational excitons can only
delocalize over two strands rather than 3 or more, which
is necessary to obtain a lower b-sheet vibrational frequency.
The similarity in the sequence between the rat amylin
and the human amylin proteins at the N-terminus as well
as the similarity of their a-helical structures suggest that
the a-helical secondary structure at the N-terminus might
be important for the hormonal activity of the protein. We
believe that the difference in the aggregation propensity
for the rat and human amylin proteins is determined by
how the residues near the C-terminus affect the stability of
the a-helical segment. In the case of the human amylin
protein, the interaction of the residues 24–28 near the
C-terminus with the a-helical segment of the protein makes
the a-helical structure less stable, causing it to misfold into a
b-hairpin. In the rat protein, the presence of proline residues
in this domain prevents such unwanted interactions, leading
to a more stable native state.
It is of interest to note that the Amyloid b polypeptide is
thought to exhibit an aggregation mechanism (and fibril
structure)similartothatofthehumanamylinprotein.Recent
NMR experiments have shown that the intermediates along
the aggregation pathway of the amyloid b peptide include
intramolecular b hairpins, similar to those observed in our
simulations (66). Hence we believe that, if our predictions
are correct, similar experiments should detect b-hairpin
structures in hIAPP (see Fig. 4 a and (67)). Also, note that
the turn region in the b-hairpin conformation presented
here for hIAPP coincides with the turn region proposed by
Luca et al. (16) for fully grown amylin aggregates.
Having established the existence of b-hairpin intermedi-
ates, we proceeded to examine their aggregation propensity.
To that end, we conducted 10 independent molecular
dynamics simulations starting from different preequilibrated
initial conditions (different Cartesian coordinates and
momenta). For clarity, representative results from only three
of those 100-ns MD trajectories are shown in Fig. 4. The
remaining trajectories do not show any qualitative differ-
ences. For these calculations, two b-hairpin conformers
were placed at a random orientation at distance ranging
from 40 to 70 A˚between their centers of mass. After an
initial equilibration period during which the separation of
the peptides was constrained, the aggregation of the two
molecules was followed (Fig. 4 a shows representative
configurations of the molecule along the aggregation
pathway). Fig. 4 b depicts the conformational variables
used here to characterize dimerization. Fig. 4 c shows
that, regardless of initial orientation, after a period of time
in the range of 20–60 ns, the molecules approach each other
to form a dimer. Fig. 4 d, which shows the relative angles
between the two hairpins as a function of time, shows that
in some cases the approach trajectory can be relatively
convoluted, with multiple twists and turns, but always
ends up in the dimerized state. Fig. 4 e shows the structural
evolution of the two hairpins and the dimer as a function of
time for one of the 10 independent trajectories considered
here (results for all trajectories exhibit the same qualitative
features). Previous studies of the aggregation pathway for
other amyloidogenic peptides indicate that the molecules
aggregate by first unfolding into a random coil (hydrophobic
collapse), and then gradually reorganizing into a b-sheet
aggregate (68). Our results show that the human amylin
peptide exhibits a high secondary structure content through-
out the aggregation pathway. Fig. 4 f shows the number of
main chain intrastrand and interstrand hydrogen bonds.
Our results indicate that, after ~20–30 ns, the two peptides
start interacting through formation of contacts between the
two chains. As shown in the figure, intrastrand hydrogen
bonds aregradually replaced by interstrand hydrogen bonds,
leading to formation of a stable dimer. The entire dimer
formation process requires a period of time in the range of
tens of nanoseconds. Additional simulations (not shown
here) also suggest that the b-hairpin conformer is able to
1550 16001650
1700
ω (cm-1)
0
0.2
0.4
0.6
0.8
1
I(ω)
α-helix
β-hairpin
random coil
weighted average
experiment
FIGURE 3
amide I stretch region for the human amylin protein at 25?C. Also shown
are line shapes for the individual conformations weighted by their relative
probabilities. The peptide concentration is 0.25 mM.
Theoretical (total) and experimental IR line shapes in the
Biophysical Journal 99(7) 2208–2216
Structure of Human Amylin Peptide 2213

Page 7

facilitate the transformation of a-helical states into addi-
tional b hairpins. The results shown in Fig. 4 provide
insights into the mechanism through which dimerization
occurs in human amylin, and they also provide initial
reactive paths that we are currently pursuing in additional
calculations aimed at characterizing the reaction coordinate
for amylin dimerization. The results of such calculations
will be presented in the future. Note here that recent work
by Rivera et al. (69) has also used simulations to examine
the dimerization of amylin. However, because those calcula-
tions were performed on fragments of the full peptide (resi-
dues 20–29), it is difficult to make a comparison to the
results presented above.
Recent experiments by Engel et al. (39) for hIAPP with
phospholipid monolayers indicate that the protein inserts
into such monolayers as a monomer. Furthermore, they
suggest that insertion occurs at the N-terminus. More-recent
NMR observations by Patil et al. (36) on sodium dodecyl
sulfate micelles also indicate that the N-terminus of hIAPP
adopts an a-helical state and inserts into the micelles. It is
knownthatthe ability ofaproteintoinsertintoamonolayers
or a bilayer is influenced by the secondary structure in
solution state. The a-helical segment on the N-terminus,
as predicted in our work, would promote its insertion into
monolayers and micelles. Note that when the amylin protein
is in an aggregated state, it would lose the a-helical
secondary structure, thereby making the insertion of the pro-
tein less favorable, as also observed by Engel et al. (39) in
their work.
CONCLUSIONS
We have presented a first, to our knowledge, atomistic scale
description of the full-length human amylin protein in
explicit water. We predict that the protein adopts three stable
conformations, one of which is a b-hairpin. The respective
abundance of each conformer is determined from precise
thermodynamic integration simulations. The results of
simulations are consistent with FTIR measurements, which
exhibit a good level of agreement with theory. Such agree-
ment is worth emphasizing in view of the many levels of
theory that have gone into our methods, and suggests that
one- and two-dimensional IR spectra can provide detailed
structural information that is not accessible in other experi-
mental approaches.
Our results also show that, despite a high degree of
sequence homology, there are fundamental differences in
the structures of the rat and the human amylin protein in
solution. The folded state of the rat amylin protein contains
an helical segment spanning residues 7–17. The human
amylin protein adopts two stable conformations with signif-
icant secondary structure: an a-helical conformation with a
short b-sheet near the C-terminus, and a full b-hairpin
conformation. The thermodynamic integration simulations
indicate that, at room temperature, the b-hairpin conforma-
tion is slightly more stable than the a-helical state.
These structural differences are particularly important in
that, in contrast to its rat counterpart, the human protein
forms amyloid fibrils. We concur with the proposal by
Dupuis et al. (42) that a possible pathway for fibril forma-
tion involves the formation and aggregation of monomeric
b-hairpins, a process in which intramolecular hydrogen
bonds are exchanged in favor of intermolecular hydrogen
FIGURE 4
tative configurations along the dimerization pathway of two human amylin
peptides in their b-hairpin conformation. The two b-hairpin structures
interact by exchanging their intramolecular hydrogen bonds for intermolec-
ular hydrogen bonds, resulting in a U-shaped b-sheet dimer structure. (b)
Schematic representation of the distance and angles between two peptides
that are used to characterize their dimerization (see below). (c) Distance
(in nm) between residues 22 (the turn residues) of the two peptides as a
function of time. Results are shown for three distinct trajectories started
from three different initial configurations: (i) molecules at a distance of
5 nm, with a q angle between the two peptides of 90?(shown in black);
(ii) molecules at a distance of 6 nm, with a q angle between them of
180?(shown in red); and (iii) molecules at a distance of 6.5 nm, with a q
angle between them of 0?(shown in blue). (d) For trajectory (i), the red
dotted line shows cos(q), the angle between the normals of the two mole-
cules. (Black solid line) Cos(F), the angle between the line joining two
peptides and one normal. (e) For trajectory (i), fraction of residues (XR)
in a particular secondary structure. The secondary structure of a residue
was assigned using the DSSP (67) approach. (Solid and dotted lines)
Results for the two different peptides, respectively. (Red, blue, and green)
Residues in b-sheet, random coil, and turn, respectively. (Solid black
line) Fraction of residues exhibiting interstrand b-bridge joining two
b-sheets. (f) For trajectory (i), (blue and red lines) number of main chain
intrastrand hydrogen bonds for the two monomers; (black line) number
of main chain interstrand hydrogen bonds.
Dimerization of hIAPP monomers in solution. (a) Represen-
Biophysical Journal 99(7) 2208–2216
2214 Reddy et al.

Page 8

bonds. We note that the turn region in the b-hairpin confor-
mation proposed here coincides with the turn region
proposed by Luca et al. (16) in the fully grown amylin
aggregates.
SUPPORTING MATERIAL
Three
supplemental/S0006-3495(10)00859-3.
figuresareavailable athttp://www.biophysj.org/biophysj/
This work was supported in part by a grant (CHE-0832584) from the
National Science Foundation (NSF) to J.L.S. and M.T.Z. J.L.S also thanks
the NSF for support of this work through grant CHE-0750307. M.T.Z.
thanks the National Institutes of Health (DK79895) for support. J.J.d.P.
thanks the NSF for support of this work through the University of Wiscon-
sin Materials Research Science and Engineering Center on Nanostructured
Interfaces and grant CBET-0755730. J.J.d.P. and J.L.S. are grateful for
support from the National Institutes of Health (1R01DK088184).
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