Short polyglutamine peptide forms a high-affinity binding site for thioflavin-T at the N-terminus

Article abstract of DOI:10.1039/c2ob07157f

Thioflavin-T is one of the most important amyloid specific dyes and has been used for more than 50 years; however, the molecular mechanism of staining is still not understood. Chemically synthesized short polyglutamine peptides (Q_n, n = 5-10) were subjected to the thioflavin-T (ThT) staining assay. It was found that the minimum Q_n peptide that stained positive to ThT was Q₆. Two types of ThT-binding sites, a high-affinity site (k_d1 = 0.1-0.17 μM) and a low-affinity site (k_d2 = 5.7-7.4 μM), were observed in short polyQs (n = 6-9). ¹³C{ ²H}REDOR NMR experiments were carried out to extract the local structure of ThT binding sites in Q₈ peptide aggregates by observing the intermolecular dipolar coupling between [3-Me-d₃]ThT and natural abundance Q₈ or residue-specific [1,2-¹³C₂] labeled Q₈s. ¹³C{²H}REDOR difference spectra of the [3-Me-d₃]ThT/natural abundance Q₈ (1/9) complex indicated that all of the five carbons of the glutamine residue participated in the formation of ThT-binding sites. ¹³C{²H}DQF-REDOR experiments of [3-Me-d₃]ThT/residue-specific [1,2-¹³C ₂] labeled Q₈ (1/50) complexes demonstrated that the N-terminal glutamine residue had direct contact with the ThT molecule at the high-affinity ThT-binding sites.

Full text of DOI:10.1039/c2ob07157f

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COMMUNICATION
Short polyglutamine peptide forms a high-affinity binding site for
thioflavin-T at the N-terminus†‡
Shigeru Matsuoka,*a,b Motoki Murai,a Toshio Yamazakic and Masayuki Inoue*a
Received 22nd December 2011, Accepted 23rd February 2012
DOI: 10.1039/c2ob07157f
Thioflavin-T is one of the most important amyloid specific
dyes and has been used for more than 50 years; however, the
molecular mechanism of staining is still not understood.
Chemically synthesized short polyglutamine peptides (Qn,
n = 5–10) were subjected to the thioflavin-T (ThT) staining
assay. It was found that the minimum Qn peptide that
stained positive to ThT was Q6. Two types of ThT-binding
sites, a high-affinity site (kd1 = 0.1–0.17 μM) and a low-
Fig. 1 (a) Chemical structures of thioflavin T (1) and [3-Me-d3]ThT
affinity site (kd2 = 5.7–7.4 μM), were observed in short
polyQs (n = 6–9). 13C{2H}REDOR NMR experiments were
carried out to extract the local structure of ThT binding sites
in Q8 peptide aggregates by observing the intermolecular
dipolar coupling between [3-Me-d3]ThT and natural abun-
dance Q8 or residue-specific [1,2-13C2] labeled Q8s. 13C{2H}
REDOR difference spectra of the [3-Me-d3]ThT/natural
abundance Q8 (1/9) complex indicated that all of the five
carbons of the glutamine residue participated in the for-
mation of ThT-binding sites. 13C{2H}DQF–REDOR exper-
iments of [3-Me-d3]ThT/residue-specific [1,2-13C2] labeled Q8
(1/50) complexes demonstrated that the N-terminal gluta-
mine residue had direct contact with the ThT molecule at the
high-affinity ThT-binding sites.
(2). (b) Structure of glutamine (Q) residue. The labeled positions of
[1,2-13C2]glutamine (Q*) are indicated by asterisks.
instance, more than forty human proteins are known to form
amyloid-like fibrils, and cause conformational diseases such as
Alzheimer’s, Parkinson’s, Huntington’s and prion diseases.4
Interestingly, all of these protein aggregates, which possess dis-
tinct amino acid sequences, are similarly stained by the common
amyloid-specific dyes, ThT and Congo-red.5 Therefore, the mol-
ecular recognition between these dyes and the peptides exhibits
both selectivity for aggregates and tolerance to different amino
acid sequences. To understand these peculiar binding character-
istics, it is necessary to elucidate the dye–amyloid complex
structure at atomic resolution.6
Thioflavin-T (ThT, 1, Fig. 1), a cationic derivative of benzothia-
zole aniline, was reported to be a fluorescent stain for amyloid in
1959.1 This compound has been widely used in basic research
and diagnosis of amyloid as one of the gold-standard dyes for
amyloid staining for more than 50 years.2,3 However, in spite of
its extensive application, the precise molecular mechanism of the
staining remains unclear. Insoluble amyloid, a fibrous β-sheet
rich aggregate, is formed by various types of proteins. For
It has been a significant challenge to study the dye–peptide
complex structure due to insolubility and heterogeneity of the
peptide aggregates. Furthermore, potential multiplicity of the
binding modes and low concentration of the binding sites in
fibrils make the situation even more problematic.7 Because of
these issues, only computational simulations of the dye–peptide
complex molecular structure have been reported,8 and partial
structural insight of the complex has been disclosed by several
X-ray diffraction studies of the dye–non-fibrillar protein
cocrystals.9
In this communication, we report the first observation of direct
molecular contacts between ThT and model peptide aggregates
by applying solid-state NMR spectroscopy. As a model for ThT-
stainable peptide aggregates, polyglutamine (polyQ) peptides
were employed, because the polyQ motif has an intrinsic ten-
dency toward aggregation and a correlation with polyglutamine
diseases.10
aGraduate School of Pharmaceutical Sciences, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: inoue@
mol.f.u-tokyo.ac.jp; Fax: +81-35841-0568; Tel: +81-35841-1354
bJST, ERATO, Lipid Active Structure Project, Toyonaka, Osaka 560-
0043, Japan. E-mail: matsuokas11@chem.sci.osaka-u.ac.jp;
Fax: +81-66850-6607; Tel: +81-66850-6605
cRIKEN System and Structural Biology Center, 1-7-22 Suehiro-cho,
Tsurumi-ku, Yokohama 230-0045, Japan
†This article is part of the Organic & Biomolecular Chemistry 10th
Anniversary issue.
‡Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob07157f
Although a number of polyQ model peptides were reported to
form amyloid-like fibrils,11 the threshold number of the
This journal is © The Royal Society of Chemistry 2012
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low-affinity sites (kd2 = 5.7–7.4 μM). Most importantly, the dis-
sociation constants of these binding sites were in the same order
of magnitude as those found in biological amyloids, suggesting
that the aggregates 4–7 functioned as model systems for the
structural analyses of the ThT-stained polyQ amyloids.14
Next, we turned our attention to the direct structural analysis
of the molecular contacts between ThT and Q8 (6) by applying
rotational-echo double-resonance (REDOR) NMR spec-
troscopy.15 To do so, deuterated ThT ([3-Me-d3]ThT (2)) was
designed and synthesized as a suitable probe for extraction of the
2
local structure in the sample. Indeed H has the lowest natural
abundance among NMR-active isotopes of the six most common
elements (CHONPS) in biological systems. 13C-observed 2H-
dephased REDOR (13C{2H}REDOR) experiments were utilized
to detect the proximity between 13C nuclei of 6 to the Me-d3
group of 2 by observing intermolecular 13C–2H dipolar coupling
(Fig. 3).16 13C{2H}REDOR experiments were carried out as a
Fig. 2 (a) Six short polyglutamines were synthesized as C- and N-
terminal free peptides. (b) Et2O precipitates of short polyglutamines
(3–8) were subjected to ThT-staining. The threshold residue number for
Qn to form ThT stainable aggregates was n = 6; above that, characteristic
fluorescent emission from bound-ThT was observed were performed
with 5 μM of ThT at three different concentrations of Qn (25, 50 and
100 μM) in glycine buffer solution pH 8.7 at 27 °C. Fluorescence from
bound-ThT was measured at ex440 nm and em470 nm.3
2
pair of 13C NMR spectra with H irradiation (REDOR: S) and
2
without H irradiation (full-echo: S0). The spatial approximation
2
between 13C and H was detected as the decay of the 13C signal
in the S spectrum according to the size of the restored 13C–2H
dipolar coupling, which is inversely proportional to the cube of
2
the interatomic distance between 13C and H. Therefore, NMR
Table 1 Dissociation constants and number of ThT-binding sites of Qn
peptidesa
signals of 13Cs in close proximity from 2H dephaser can be
extracted by taking the difference spectrum S0 − S (REDOR
difference, ΔS).
Peptide
kd1/μM
N1/10−3
kd2/μM
N2/10−3
4
5
6
7
0.17
0.11
0.10
0.15
20.1
19.4
21.7
25.1
6.45
5.72
6.76
7.42
77.7
65.0
86.3
82.4
First, 50 μM of 2 was mixed with 100 μM of Q8 (6) in a ratio
of 1/9 mol/mol (ThT/peptide) in aqueous buffer, and then the
solution was lyophilized. Under these conditions, both high-
affinity and low-affinity sites were occupied. The resulting
powder sample was used in the solid state NMR measurement.
The 13C NMR spectrum of the 2/6 complex was measured as a
full echo S0 spectrum shown in Fig. 3, bottom. Resonances
arising from natural abundance 13C gave five resolved peaks of
the glutamine unit (Cα, Cβ, Cδ, Cγ, and CvO); however, all
eight residues were overlapped. 13C chemical shifts of CvO and
Cα corresponded to those in a β-sheet structure.17
a Two distinct binding sites were observed with higher affinity (Kd1, N1)
and lower affinity (Kd2, N2). N is the number of binding sites for one Qn
molecule.
glutamine residues required for formation of aggregates was not
determined. To simplify the structural analysis of the complex,
we decided to first elucidate the minimum ThT-stainable polyQ
lengths. Accordingly, a series of short polyQs, 3–8 (Fig. 2a),
were synthesized by Fmoc solid phase peptide synthesis, and the
products were treated with diethylether to precipitate the corre-
sponding aggregates. The aggregates were insoluble in both
water and various organic solvents, except for Q5 (3), which was
water soluble. X-ray diffraction studies of the aggregates clearly
indicated that Q6–Q10 (4–8) mainly contained β-sheets, the
typical secondary structure of amyloid fibrils.12
The formation of ThT-binding sites in Q5–Q10 (3–8) was eval-
uated by the ThT fluorescent staining assay (Fig. 2b).3 Synthetic
polyQs longer than six residues (4–8) possessed considerably
enhanced ThT fluorescence with excitation and emission
maxima at 440 nm and 470 nm respectively, at the three different
concentrations studied. The results indicated formation of ThT-
binding sites in the insoluble aggregates of 4–8. According to
Scatcherd analysis of 4–7,13 similar high- and low-affinity ThT
binding sites were found in all the aggregates (Table 1).
Numbers and the affinities of the two binding sites were inconse-
quential to the length of the peptides. The high-affinity sites (kd1
= 0.1–0.17 μM) were composed of approximately 50 peptides,
while ca. 12 peptides were necessary for the formation of the
Fig. 3 13C{2H}REDOR spectra of [3-Me-d3]ThT (2)/Q8 (6) (1/9 mol/
mol) complex. Full-echo 13C NMR spectrum (S0) is shown at the
bottom and REDOR difference (ΔS) is shown on the top with 20× mag-
nification. Spectra were recorded with a dephasing time of 9.6 ms (80
rotor cycles) at a MAS speed of 8333 Hz. The number of scans
was175 000. Signals arisen from ThT are marked by filled squares. The
spinning side bands are marked with an asterisk.
5788 | Org. Biomol. Chem., 2012, 10, 5787–5790
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The 13C{2H}REDOR difference spectrum ΔS of 2/6 complex
is shown in Fig. 3, top. For efficient selective observation of 13
C
nuclei adjacent to the CD3 group, 9.6 ms of dephasing time was
applied to obtain the maximum REDOR dephasing for 13C
nuclei within 3.1 Å from the center of three deuterons.18 All five
13Cs gave ΔS signals in the difference spectrum with intensities
over 2% of S0, which indicated that the five carbon atoms of the
glutamine unit were in proximity to 2 through participation in
the high- and/or low-affinity binding sites. However, determi-
nation of the specific residue that neighbored 2 was not possible
due to the signal overlap among the eight glutamine residues
of 6.
To assign the ThT-contacting residue, a series of eight residue-
specific 13C2-labeled Q8s 9–16 were prepared by incorporating
the asymmetrically synthesized [1,2-13C2]Gln (Fig. 1b and 4),
and subjected to the solid-state NMR study. The extraction of
13C NMR signals from a particular residue was achieved by
combination of residue-specific labeling by [1,2-13C2]Gln and
13C–13C pair selection by double-quantum filtering (DQF) to
suppress the large background signals arising from natural abun-
dance 13Cs of the other seven Gln residues.19 For specific analy-
sis of the local structure of the high-affinity sites, the
concentration of 2 was lowered in these experiments. Namely,
residue specific labeled Q8 peptides 9–16 were incubated with 2
at a ratio of 1/50 mol/mol (ThT/peptide), where ThT molecules
and the number of high-affinity binding sites were equivalent,
and then frozen with liquid nitrogen to be lyophilized.
Fig. 5 (a) 13C CP-MAS spectrum of [3-Me-d3]ThT (2)/Q*Q7 (9) (1/
50 mol/mol). (b) 13C{2H}DQF–REDOR spectra of the 2/9 (1/50 mol/
mol) complex. DQF full-echo 13C NMR spectrum (S0) is shown at the
bottom and REDOR difference (ΔS) is shown on the top with 20× mag-
nification. Spectra were recorded with a dephasing time of 9.6 ms (80
rotor cycles) at a MAS speed of 8333 Hz. The number of scans was
100 000. The spinning side bands are marked with an asterisk.
The in-phase DQF spectra were measured for each of 9–16.
Naturally abundant 13C signals from the side chain Cβ, Cγ, and
Cδ were sufficiently suppressed in the DQF spectra; conse-
quently, only the signals from the main chain 13Cα and 13CvO
of [1,2-13C2]Gln were selectively observed (Fig. 4 and 5).20
With the residue-selected DQF spectra of 9–16 in hand, we
undertook assignment of the glutamine residue contacting the 2H
by 13C{2H}DQF–REDOR experiments (Fig. 5b).21 Intriguingly,
only 13C DQF signals of 2/9, which has the 13Cα–13CvO unit at
the first residue from the N-terminus, exhibited a detectable
REDOR difference in ΔS when dephased by the 3-CD3 group of
2. Thus, in terms of proximity to the CD3 group of ThT, the N-
terminal glutamine residue contacts 2. Since the chemical shifts
Fig. 6 (a) Ratio of ΔS/S0 between two dephasing times, 4.8 ms and
9.6 ms, plotted against the C–CD3 interatomic distance (rCD). The solid
lines show the simulated values for the shortest (red) and the longest
(blue) case.22 The open square and open circle show the experimental
results of 2/9 (1/9 mol/mol) complex obtained for CvO (0.65) and Cα
(0.55) respectively. (b) Schematic diagram of ThT–Q8 aggregate
complex structure. The Q8 aggregate is shown as an antiparallel β-sheet
(white). The N-terminal Q residue is presented in atom-coded colors (C:
grey, H: white, O: red, N: blue, 13C: green). According to REDOR dis-
tance constraints,22 ThT (orange) was placed to avoid steric repulsions
with the Q8 aggregate.
of 13Cα–13CvO in the ΔS spectrum corresponded to a β-sheet
structure, the first glutamine residue of Q8 forming the ThT-
binding sites also participated in the β-sheet structure.
To gain a quantitative understanding of interatomic distances
between the CD3 group of 2 and the 13Cα–13CvO unit of 9,
REDOR difference spectra were measured at two different
dephasing times, 4.8 ms and 9.6 ms, for the 2/9 complex.
Fig. 6a shows a plot of R4.8/9.6, the ratio of ΔS/S0 between the
two dephasing times, against the C–CD3 distance. When the
Fig. 4 13C DQF spectra of residue-specific [1,2-13C2] labeled Q8s. The
labeled positions are shown on the left and the DQF spectra are shown
on the right. The 13C–13C bilinear term was evolved and refocused for
9.6 ms (80 Tr).
This journal is © The Royal Society of Chemistry 2012
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Biophys. Acta, 2010, 1804, 1405–1412; (c) M. Groenning, J. Chem.
Biol., 2010, 3, 1–18.
7 H. Levine III, Amyloid, 2005, 12, 5–14.
C–CD3 distance (rCD) is longer than the van der Waals contact
range (>3.12 Å) and R4.8/9.6 is less than 1, the value of R4.8/9.6
has a one-to-one correspondence with the C–CD3 distance and
thus can be used for distance estimation. Assuming a single dis-
tance component, the C–CD3 distances were deduced to be
3.6–4.0 Å and 3.9–4.3 Å for CvO (R4.8/9.6 = 0.65) and Cα
(R4.8/9.6 = 0.55), respectively. Using the distance constraints from
the second residue (no detectable REDOR dephasing for 2/10)
as well as avoiding contact within the van der Waals surfaces of
2 and Q8, the possible geometry of CD3 in relation to the poly-
glutamine residue was estimated as shown in Fig. 6b.22 This
model indicated that the high-affinity ThT-binding site in the Q8
aggregate was located at the gap in the β-sheet structure where
the N-terminal Q residue has a hydrogen bonding partner only
on one face. The geometry of the CD3 group implied the pres-
ence of a CH3⋯OvC interaction between the 3-methyl group of
ThT and the main chain carbonyl group of the N-terminal Q
residue, which may be one of the possible interactions stabilizing
the ThT–Q8 complex at the high-affinity binding site.23
In conclusion, we synthesized various short polyQs (Q6–Q10),
determined the minimum peptide stained by the amyloid specific
dye ThT to be Q6, and observed the intermolecular contacts
between ThT and Q8 by REDOR NMR spectroscopy. The short
polyglutamine aggregates possessed β-sheet rich structures, and
their high- and low-affinity binding sites corresponded to the
same order of binding affinity as those formed in natural amy-
loids. Eight residue-specific labeled Q8(n-[1,2-13C2]Gln) pep-
tides were synthesized and mixed with [3-Me-d3]ThT for the
solid-state NMR measurements. 13C{2H}DQF–REDOR exper-
iments of these residue specific labeled samples successfully
resulted in the assignment of the N-terminal residue that was in
direct contact with ThT at the high-affinity binding site.
Although the peptides used in this study were a simplified
model, the strategy employed here to obtain precise local struc-
tural information should be generally applicable to binding sites
in other amyloids, and studies along this line are currently
ongoing.
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12 The characteristic 4.8 Å reflection, which is attributed to the strand
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shown in the ESI as Fig. S2.‡ We thank Dr Yuya Koike (Radioisotope
Center, The University of Tokyo) for help in measurement of X-ray
diffraction.
13 Experimental results were shown as a Scatcherdplot (Fig. S3) in the
ESI.‡.
14 The affinity of ThT towards various types of amyloide has been
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shown in the ESI as Fig. S9.‡.
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those observed for the β-sheet structure, except for the carboxylic acid
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22 Detailed distance restraints are shown in the ESI.‡.
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This research was financially supported by the Funding
Program for Next Generation World-Leading Researchers (M.I.)
and Grant-in-Aids for Young Scientists (start up) (S.M.).
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This journal is © The Royal Society of Chemistry 2012