A short water-soluble self-assembling peptide forms amyloid-like fibrils

Article abstract of DOI:10.1039/b607657b

A water-soluble tripeptide Val-Ile-Ala (VIA) 1, bearing sequence identity with the C-terminal portion of the Alzheimer Aβ-peptide (Aβ_40-42), self-assembles, in crystalline form, to produce an intermolecularly hydrogen bonded supramolecular β-sheet structure which self-associates to form straight, unbranched nanofibrils exhibiting amyloid-like behavior; in contrast, the synthetic tripeptide Ala-Val-Ile (AVI) 2 self-assembles to produce a β-sheet structure that forms branched nanofibrils which do not show any characteristic features of amyloid-like fibrils. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b607657b

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A short water-soluble self-assembling peptide forms amyloid-like
fibrils{
a
Sudipta Ray, Apurba K. Das, Michael G. B. Drew and Arindam Banerjee*
a
b
a
Received (in Cambridge, UK) 31st May 2006, Accepted 8th August 2006
First published as an Advance Article on the web 30th August 2006
DOI: 10.1039/b607657b
A water-soluble tripeptide Val–Ile–Ala (VIA) 1, bearing
sequence identity with the C-terminal portion of the
Alzheimer Ab-peptide (Ab40–42), self-assembles, in crystalline
form, to produce an intermolecularly hydrogen bonded
supramolecular b-sheet structure which self-associates to form
straight, unbranched nanofibrils exhibiting amyloid-like beha-
vior; in contrast, the synthetic tripeptide Ala–Val–Ile (AVI) 2
self-assembles to produce a b-sheet structure that forms
branched nanofibrils which do not show any characteristic
features of amyloid-like fibrils.
contribution in investigating the residue-specific effect on the
propensity of a given hexapeptide sequence to form amyloid
9
fibrils. Lynn et al. have also made important contributions to the
structural characterization of amyloid fibrils using solid-state
10
NMR techniques.
Our group has been studying the self-association of terminally
protected short model peptide structures into supramolecular
b-sheets; these structures form amyloid-like fibrils upon further
1
1
self-association. However, we present here the self-association, in
crystalline form, of a water-soluble tripeptide, comprised of the Ab
peptide residue 40–42 (VIA), peptide 1 (Fig. 1b), which forms an
intermolecularly hydrogen bonded supramolecular b-sheet struc-
ture. This tripeptide also forms straight, unbranched nanofibrils
which exhibit amyloid-like behavior. Another synthetic tripeptide
2 (AVI), having the same amino acid composition but a different
sequence from that of the tripeptide 1 (Fig. 1c), has been
synthesized, purified, characterized and studied to establish
whether the amyloid-like fibrillation is sequence specific or not.
Preliminary investigations, regarding the secondary structures of
peptides 1 and 2, have been carried out using solid state FT-IR
spectroscopy. FT-IR spectra of both peptides are characterized by
Alzheimer’s disease (AD) is one of the most prevalent and well
characterized fatal neurodegenerative diseases, in which self-
aggregated b-sheet rich protein fragments deposit in specific
1
regions of the human brain as amyloid fibrils. The amyloid fibrils
in AD generally consist of a b-amyloid protein (Ab1–42), which is
2
produced from a larger amyloid precursor protein (APP) by
proteolytic cleavage. There are three specific regions in the Ab1–42
peptide: (a) a hydrophilic N-terminus region consisting of residues
1–16, (b) a central hydrophobic stretch consisting of residues 17–21
and (c) a long hydrophobic C-terminus consisting of residues 29–
3
2. It has been observed that the C-terminal sequence is critical
21
4
well-defined CO-stretching bands at around 1633–1654 cm and
21
the NH-stretching band at around 3273–3303 cm , typical for
4
for amyloid fibril formation. The self-aggregated amyloid fibrils
5a
5b
are generally formed from parallel or antiparallel cross b-sheet
structures. Due to their low solubility and non-crystallinity, the
structure determination of amyloid fibrils, obtained from the full
length Ab protein or its larger segment, becomes almost impossible
using single crystal X-ray diffraction studies. Meticulous under-
standing of the self-association of amyloid fibrils is absolutely
necessary to obtain a detailed knowledge concerning b-sheet
aggregation and to develop a therapeutic agent against amyloid
diseases in the future. Solid state NMR spectroscopy is also a
powerful technique for elucidating the structures of non-crystalline
fibrous materials like amyloid fibers. Earlier solid state NMR
studies from Lansbury’s group, regarding the amyloid fibrils
obtained from a self-assembling peptide Ab34–42, indicate that the
intermolecularly hydrogen bonded b-sheet structures in the solid
1
2
state (ESI Fig. S5{). Moreover, peptide 1 shows a shoulder at
1685 cm indicating the formation of an antiparallel b-sheet
21
structure. Furthermore, the NH-bending frequencies of peptides 1
2
1
21
and 2 appear at 1540 cm and 1556 cm respectively, suggesting
13
the formation of a b-sheet structure.
Preliminary analysis of the conformational features of peptide 1
has been further supported by single-crystal X-ray diffraction
1
4
studies. Colorless needle-shaped single crystals, suitable for X-ray
analysis, have been obtained from slow evaporation, at room
6,7
amyloid fibrils form a pleated antiparallel b-sheet structure.
Recent studies have established that amyloid fibrils formed from
the residue Ab11–25 at different pHs also exhibit the antiparallel
8
b-sheet structure. Serrano and co-workers have made a pioneering
a
Department of Biological Chemistry, Indian Association for the
Cultivation of Science, Jadavpur, Kolkata, India.
E-mail: bcab@mahendra.iacs.res.in; Fax: +91-33-2473-2805
School of Chemistry, The University of Reading, Whiteknights,
Reading, UK RG6 6AD
{ Electronic supplementary information (ESI) available: Experimental
procedures, full spectroscopic data for all new compounds and CIF for
b
Fig. 1 (a) Amino acid sequence of Ab1–42 (isolated from an amyloid
plaque core from an AD brain), (b) sequence and chemical structure of
model amyloid peptide Ab40–42 (peptide 1) and (c) sequence and chemical
structure of peptide 2, which has a different sequence.
peptide 1. See DOI: 10.1039/b607657b
4
230 | Chem. Commun., 2006, 4230–4232
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temperature, of a water–ethanol (98 : 2) solution of peptide 1
200 mg per 30 mL). The molecular conformation of peptide 1
ESI Fig. S6{) illustrates that there are four molecules (designated
carboxylic anions play an important role in forming the
supramolecular b-sheet structure in the solid state without solvent.
FT-IR data also support the zwitterionic nature of the peptide 1 in
the solid state (ESI Fig. S5).{ There are two intermolecular
hydrogen bonds, namely N8C–H O6D and N8D–H O6C, that
are responsible for connecting the individual molecules (C and D)
to form the dimer of peptide 1 along the crystallographic a axis
(Fig. 2a). Four intermolecular hydrogen bonds, namely N5C–
(
(
as A, B, C and D) in the asymmetric unit. The molecules A and B
are joined together by four intermolecular hydrogen bonds and the
other two molecules, C and D, are joined together by two
intermolecular hydrogen bonds between amide CLO and NH
functionalities to form two stable molecular dimers in the
asymmetric unit. Backbone torsion angles of each molecule (A,
B, C and D) of peptide 1 fall mostly within the extended region of
…
…
…
… … …
H
O9D, N5D–H O9C, N10D–H O2C and N10C–H O2D,
are involved in joining the individual dimers of peptide 1 to form
the monolayer b-sheet structure along the crystallographic a axis.
There are four intermolecular hydrogen bonds, namely N10C–
1
5
the Ramachandran diagram (ESI Table 1{). Thus, peptide 1
provides an overall extended backbone structure for each molecule
present in the asymmetric unit. There are four intermolecular
…
…
…
H
H
O1B, N10C–H O2B, N10D–H O1A and N10D–
O2A, that are responsible for connecting to the other
…
…
…
hydrogen bonds, namely N10B–H O2A, N5A–H O9B, N5B–
…
…
O9A and N10A–H O2B, that are responsible for connecting
H
molecules, A and B, of peptide 1 to form a corrugated b-sheet
structure (Fig. 2b).
the individual molecules (A and B) to form the dimer of peptide 1
along the crystallographic a axis (Fig. 2a). The molecular duplex
formed by the molecules A and B self-assembles, via intermole-
cular hydrogen bonds and other non-covalent interactions, to form
an infinite antiparallel b-sheet assemblage in the crystal along the
crystallographic a direction. Another two intermolecular hydrogen
Both peptides, 1 and 2, self-assemble to form nanofibrillar
structures (Fig. 3). However, their morphologies are different.
Transmission electron microscopic (TEM) studies of peptide 1
reveal that it has a straight, unbranched nanofibrillar morphology
with a diameter of 5–10 nm (Fig. 3a), while the fibrils formed by
peptide 2 are small, branched fibrils with a diameter of 10–20 nm
(Fig. 3b), indicating their non-amyloidogenic fibrillar nature.
Nanofibrils obtained from these reported peptides, 1 and 2, have
been examined for staining with a physiological dye, Congo red,
and viewed through a cross polarizer, under a polarized
microscope, to look for the typical birefringence of amyloid fibrils.
Peptide 1 binds with Congo red and exhibits a typical green gold
birefringence when viewed through the cross polarizer, indicating
amyloid-like behavior (ESI Fig. S7a{). However, the fibrils
obtained from peptide 2 do not show any characterized
birefringence typical for amyloid fibrils, after being stained with
the dye Congo red and viewed through the cross polarizer (ESI
Fig. S7b{).
…
…
bonds, N8A–H O6B and N8B–H O6A, are involved in joining
the individual dimers of peptide 1 to form the monolayer b-sheet
structure along the crystallographic a axis. These individual b-sheet
columns are themselves regularly stacked, via van der Waals
interactions, to form a complex quaternary supramolecular b-sheet
structure. The hydrogen bonding parameters of peptide 1 are listed
in the ESI Table 2{. There are four intermolecular hydrogen
…
…
bonds, namely N10A–H O2D, N10A–H O1D, N10B–
…
…
O2C and N10B–H O1C, that are responsible for joining
H
the other molecules, C and D, of peptide 1 to form the pleated
b-sheet structure. The molecular duplex formed by the molecules C
and D also self-assembles, via intermolecular hydrogen bonding
and other non-covalent interactions, to form an infinite antiparallel
b-sheet assemblage in the crystal along the crystallographic a
direction (Fig. 2a).
The aged solution of tripeptide 1 fibrils exhibited double minima
2
1
at 1633 and 1650 cm (ESI Fig. S9{). This amide I FT-IR
spectrum is consistent with an antiparallel b-sheet structure.
9,16
These individual b-sheet columns are themselves regularly
stacked, via van der Waals interactions, to form a complex
quaternary supramolecular b-sheet structure. Electrostatic interac-
tions and hydrogen bonding of the ammonium groups and
2
1
The amide I band at 1634 cm , observed with the aged solution
of tripeptide 2 fibrils, is typical for the formation of a b-sheet
9,16
structure. From the CD spectral data (ESI Fig. S10{) it may be
concluded that, at room temperature, the aged solution of peptide
2
fibrils is dominated more by some type of unstructured or
random structure than by that of the aged solution of peptide 1
fibrils.
Like Congo red dye, thioflavin T (ThT) is another amyloid-
specific dye. Fig. 4 shows the enhancement of the fluorescence of
Fig. 2 (a) Packing diagram of peptide 1 along the crystallographic a
direction, illustrating intermolecular hydrogen bonding in the solid state
and the formation of continuous antiparallel b-sheet columns. (b) Higher
order packing of peptide 1 forming quaternary b-sheet structures.
Hydrogen bonds are shown as dotted lines. Only hydrogen bonded H
atoms are shown for clarity.
Fig. 3 TEM images of the fibril formation of (a) peptide 1 and
(b) peptide 2. The peptides were dissolved in distilled water at pH 7
21
(c = 1 mg mL ) and incubated at 37 uC for 7 days.
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Chem. Commun., 2006, 4230–4232 | 4231

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of a peptide in amyloid-like fibril formation. The effect of
temperature and the pH stability of the fibres will be explored in
the near future.
This research is supported by a grant from the Department of
Science and Technology (DST), India (Project No. SR/S5/OC-29/
2003). We thank the EPSRC and the University of Reading, UK,
for funds for the X-Calibur System. S. Ray and A. K. Das wish to
acknowledge the CSIR, New Delhi, India, for financial assistance.
We gratefully acknowledge the Nanoscience and Technology
initiative of DST, Govt of India, for using their TEM facility.
Fig. 4 Thioflavin T (ThT) binding assay for peptide 1.
Notes and references
1
(a) K. S. Kosik, J. Cell Biol., 1994, 127, 1501–1514; (b) K. S. Kosik,
Science, 1992, 256, 780–783.
ThT after binding with the nanofibrils obtained from peptide 1.
This enhancement is more than three times higher for a solution of
2
(a) G. G. Glenner and C. W. Wong, Biochem. Biophys. Res. Commun.,
1984, 122, 1131–1135; (b) C. L. Masters, G. Simms, N. A. Weinmann,
G. Multhaup, B. L. McDonald and K. Beyreuther, Proc. Natl. Acad.
Sci. U. S. A., 1985, 82, 4245–4249; (c) J. Kang, H. G. Lemaire,
A. Unterbeck, J. M. Salbaum, C. L. Masters, K. H. Grzeschik,
G. Multhaup, K. Beyreuther and B. Muller-Hill, Nature, 1987, 325,
733–736; (d) F. Prelli, E. Castano, G. G. Glenner and B. Frangione,
J. Neurochem., 1988, 51, 648–651.
2
1
peptide 1 (0.4 mg mL ) fibrils containing ThT and incubated for
one week than that observed for ThT alone. Peptide 2 does not
show any significant binding and fluorescence enhancement with
ThT, indicating its non-amyloid character.
FT-IR studies clearly suggest that both peptides self-assemble to
form hydrogen bonded supramolecular b-sheet structures in the
solid state and in fibrils. The crystal structure of peptide 1 further
supports the formation of an antiparallel b-sheet structure using
multiple hydrogen bonds. Interestingly, peptide 1 forms amyloid-
like fibrils as is evident from a typical green gold birefringence after
staining with the physiological dye Congo red and the enhance-
ment of fluorescence upon binding with another specific dye,
thioflavin T. The peptide 1 also forms straight, unbranched
nanofibrils. Both peptides 1 and 2 have the same amino acid
compositions; however, peptide 2 has an amino acid sequence that
is different from peptide 1. Peptide 2 also self-assembles to form
nanofibrillar structures but its morphology is entirely different
3
T. S. Burkoth, T. L. S. Benzinger, V. Urban, D. M. Morgan,
D. M. Gregory, P. Thiyagarajan, R. E. Botto, S. C. Meredith and
D. G. Lynn, J. Am. Chem. Soc., 2000, 122, 7883–7889.
4 J. T. Jarrett, E. P. Berger and P. T. Lansbury, Biochemistry, 1993, 32,
693–4697.
5
4
(a) L. Hou and M. G. Zagorski, Biophys. J., 2004, 86, 1–2; (b)
D. J. Gordon, J. J. Balbach, R. Tycko and S. C. Meredith, Biophys. J.,
2004, 86, 428–434.
P. T. Lansbury, P. R. Costa, J. M. Griffiths, E. J. Simon, M. Auger,
K. J. Halverson, D. A. Kocisko, Z. S. Hendsch, T. T. Ashburn,
R. G. S. Spencer, B. Tidor and R. G. Griffin, Nat. Struct. Biol., 1995, 2,
6
990–998.
7 J. M. Griffiths, T. T. Ashburn, M. Auger, P. R. Costa, R. G. Griffin
and P. T. Lansbury, J. Am. Chem. Soc., 1995, 117, 3539–3546.
8
9
A. T. Petkova, G. Buntkowsky, F. Dyda, R. D. Leapman, W.-M. Yau
and R. Tycko, J. Mol. Biol., 2004, 335, 247–260.
M. L. de la Paz, K. Goldie, J. Zurdo, E. Lacroix, C. M. Dobson,
A. Hoenger and L. Serrano, Proc. Natl. Acad. Sci. U. S. A., 2002, 99,
(branched and small) from that observed for peptide 1. Fibrils
obtained from peptide 2 fail to exhibit amyloid-like behavior.
Previous reports have shown the formation of amyloid fibrils from
short water-soluble peptides like pentapeptide or tetrapeptide
16052–16057.
1
6
10 (a) T. L. S. Benzinger, D. M. Gregory, T. S. Burkoth, H. Miller-Auer,
D. G. Lynn, R. E. Botto and S. C. Meredith, Proc. Natl. Acad. Sci.
U. S. A., 1998, 95, 13407–13412; (b) T. L. S. Benzinger, D. M. Gregory,
T. S. Burkoth, H. Miller-Auer, D. G. Lynn, R. E. Botto and
S. C. Meredith, Biochemistry, 2000, 39, 3491–3499.
segments of real amyloidogenic proteins/peptides and also from
9
de novo designed hexapeptides. Herein, our present study clearly
demonstrates the formation of amyloid-like fibrils from a water-
soluble tripeptide segment, comprised of the C-terminal part of
Ab1–42 (i.e. Ab40–42). To the best of our knowledge, this is the
shortest peptide segment that not only forms amyloid-like fibrils,
but also produces needle-shaped crystals perfectly suitable for
single crystal X-ray diffraction studies in order to determine its
structure and intermolecular arrangements at atomic resolution.
This study clearly demonstrates that a short water-soluble
tripeptide, comprised of the three C-terminal amino acids of
Ab1–42 (i.e. Ab40–42), self-assembles to form a supramolecular
b-sheet structure in crystals and that this tripeptide also forms
amyloid-like fibrils from an aqueous solution under the proper
conditions. The molecular arrangement of the self-association of
this fibril forming peptide, is clearly understood using single crystal
X-ray diffraction studies. This can help to understand how self-
association occurs in amyloid-like fibrillation using short peptide
segments. Interestingly, peptide 2, which has a different amino acid
sequence but the same amino acid composition as peptide 1, fails
to form amyloid-like fibrils, indicating the sequence specific nature
1
1 (a) S. K. Maji, M. G. B. Drew and A. Banerjee, Chem. Commun., 2001,
446–1447; (b) A. Banerjee, S. K. Maji, M. G. B. Drew, D. Haldar and
A. Banerjee, Tetrahedron Lett., 2003, 44, 6741–6744.
2 Y. Mazor, S. Gilead, I. Benhar and E. Gazit, J. Mol. Biol., 2002, 322,
013–1024.
3 V. Moretto, M. Crisma, G. M. Bonora, C. Toniolo, H. Balaram and
P. Balaram, Macromolecules, 1989, 22, 2939–2944.
14 Crystal data for peptide 1: 4(C14 )?CF
18.5), FW = 1342.61, triclinic, space group P
1
1
1
1
H
27
N
3
O
4
3
CO
2
H?0.5C
2
H
5
OH,
, a =
.6224(5), b = 14.0220(8), c = 16.2566(9) A, a = 101.369(5), b =
(
9
C
59
H
112
F
3
N
12
O
1
˚
23
21
1
05.131(5), c = 90.220(4)u, Z = 1, dcalc = 1.076 g cm , m = 0.084 mm
intensity data for peptide 1 were collected with CuKa radiation at 100 K.
int = 0.0348. The final R values were R1 = 0.1266 and wR2 = 0.3327
,
R
for 7352 data (I . 2s(I)) for peptide 1. The high R value was due to the
fact that the crystal was twinned. The asymmetric unit contains
4
molecules of peptide 1 together with one molecule of trifluoroacetic
acid and 0.5 molecules of ethanol. CCDC 609564. For crystallographic
data in CIF or other electronic format see DOI: 10.1039/b607657b.
1
5 G. N. Ramachandran and V. Sasisekharan, Adv. Protein Chem., 1968,
3, 284–438.
6 M. Reches, Y. Porat and E. Gazit, J. Biol. Chem., 2002, 277,
35475–35480.
2
1
4
232 | Chem. Commun., 2006, 4230–4232
This journal is ß The Royal Society of Chemistry 2006