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48
Experimental Methods
Sample preparation
The peptides for this study were custom synthesized
from M/s USV peptides and purity of >95% was conA
Figure 1: Seven hundred megahertz proton spectrum of Ab12 in phosphate buffer pH 6 at 280 K. The expanded regions are (A) amide
and aromatic protons, (B) Ca Protons and (c) remaining side-chain protons.
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Narayan et al.
Structure calculations
Structures of the peptides were calculated using the software CYANA (version no. 3.0) by including the constraints
derived from NMR. The starting structure in all the cases
was the extended form of the peptide sequence. The relative NOE cross-peak volumes manually calculated from
the ROESY spectrum were converted to approximate distance ranges and were provided as distance constraint
input file (Tables S2 and S3). A set of structures are calculated by the software, based on torsion angle dynamics
driven simulated annealing (42). If all experimental constraints are satisfied and all nonbonded atom pairs are free
of steric overlap, then a target function (f) is minimized,
which is indicative of quality of structure obtained. In our
peptides, we had a limited NOE distance and dihedral
angle () constraints, which were obtained from NMR
Table 1: NMR parameter of free Ab12 along with Al3+ metal-bound peptide (in brackets)
Chemical shift (ppm)
Resi-due
NH
D(1)
A(2)
E(3)
F(4)
R(5)
H(6)
D(7)
S(8)
G(9)
Y(10)
E(11)
V(12)
8.72
8.40
8.31
8.25
8.65
8.54
8.47
8.50
7.98
8.29
7.87
CaH
(8.73)
(8.36)
(8.29)
(8.25)
(8.62)
(8.57)
(8.47)
(8.48)
(7.98)
(8.24)
(8.00)
4.17
4.23
4.15
4.52
4.14
4.57
4.58
4.33
3.86
4.48
4.26
3.94
CbH
(4.19)
(4.24)
(4.17)
(4.52)
(4.14)
(4.57)
(4.61)
(4.33)
(3.82)
(4.48)
(4.28)
(3.98)
2.79,
1.29
1.89
2.94,
1.59,
3.07,
2.64,
3.80
CcH
2.82 (2.74,
(1.29)
(1.89)
3.00 (2.95,
1.68 (1.59,
3.15 (3.08,
2.71 (2.77)
(3.80)
2.83)
CdH
CeH/Others
3.03 (3.06)
7.19 (7.17)
7.14 (7.19)
7.32 (7.26)
8.51 (8.52)
6.99 (6.99)
6.71 (6.71)
JNHcaH (Hz)
5.5
6.5
7.0
6.9
7.3
6.8
6.1
6.9
7.4
8.1
(5.4)
(6.5)
(6.8)
()
(7.2)
(6.8)
(5.6)
(5.9)
()
(6.7)
(7.7)
50
recordings. Although amide proton temperature coefficients were obtained, it could not be used to fix appropriate hydrogen bond distances because of lack of
knowledge of the bonded partner. Twenty structures were
calculated which resulted in no violations of any of the
constraints. All the and w dihedral values were also
within the allowed region in the Ramachandran Map. Typically, average RMSD values for all 20 structures (residues
312) were 0.37 0.11
A for backbone atoms and
0.94 0.21
A when all heavy atoms were considered.
Best five structures were considered in each case for
comparison.
Molecular modelling
Molecular modelling to identify the metal-binding sites
was carried out using commercial Schrodinger software.
NMR-derived CYANA structures were exported to macromodel module of the software. Couple of potential aluminium-binding site were identified near the charged residues
Figure 3: Stereo view of best five of 20 structures calculated using CYANA software by using only limited NOE distant and dihedral
constraints. Hydrogen bond constraints could not be used due to lack of identification of partner carbonyl oxygen to amide protons.
Table 2: NMR parameter of free Ab16 along with Al3+ metal-bound peptide (in brackets)
Chemical shift (ppm)
Resi-due
NH
D(1)
A(2)
E(3)
F(4)
R(5)
H(6)
D(7)
S(8)
G(9)
Y(10)
E(11)
V(12)
H(13)
H(14)
Q(15)
K(16)
8.61
8.27
8.15
8.11
8.49
8.39
8.37
8.44
7.91
8.18
7.93
8.42
8.48
8.47
8.08
CaH
()
(8.62)
(8.21)
(8.13)
(8.11)
(8.48)
(8.44)
(8.35)
(8.38)
(7.92)
(8.11)
(7.92)
(8.43)
(8.50)
(8.49)
(8.26)
4.25
4.27
4.18
4.55
4.20
4.61
4.67
4.36
3.86
4.45
4.20
3.85
4.70
4.66
4.30
4.12
CbH
(4.25)
(4.29)
(4.22)
(4.55)
(4.20)
(4.60)
(4.66)
(4.37)
(3.87)
(4.46)
(4.20)
(3.85)
(4.70)
(4.66)
(4.30)
(4.12)
CcH
2.73,
1.30
1.89
2.94,
1.64,
3.06,
2.69
3.83
2.81 (2.82)
(1.30)
(1.89)
3.03 (2.95, 3.05)
1.68 (1.62, 1.68)
3.19 (3.09, 3.19)
(2.77)
(3.83)
2.91
1.85,
1.88
3.04,
3.09,
1.96,
1.70,
(2.91)
1.91 (1.84,
(1.87)
3.17 (3.07,
3.19 (3.06,
2.07 (1.95,
1.76 (1.74,
1.92)
3.17)
3.21)
2.06)
1.80)
CdH
CeH/Others
7.16 (7.17)
3.08
7.24 (7.24)
7.26 (7.27)
7.17 (7.18)
8.55 (8.56)
7.02 (7.02)
6.73 (6.73)
7.22 (7.22)
7.20 (7.21)
8.54 (8.55)
8.52 (8.54)
2.18 (2.26)
0.76, 0.81 (0.73, 0.82)
2.34 (2.35)
1.38 (1.39)
JNHcaH (Hz)
5.8
6.7
7.1
7.1
7.2
7.1
6.3
6.5
6.8
7.2
7.9
7.7
7.3
(5.8)
(6.7)
(7.1)
()
(7.6)
(7.1)
(6.3)
()
(6.3)
()
(7.0)
(7.8)
(7.6)
(6.9)
(7.2)
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Narayan et al.
Ab16 structure
Following the method described above, the structure of
the 116 N-terminal fragment of Ab peptide {DAEFRHDSGYEVHHQK, Ab16} was also calculated. Ab16 has
four extra residues (HHQK) at C-terminus, in comparison
with Ab12. Relevant part of the TOCSY and ROESY spectra of Ab16 are given in Supporting information (S3 and
S4). The chemical shifts along with 3JNHCaH values are
provided in Table 2. The NMR spectra recorded at 303K
show a good dispersion in the amide region. Ab16 structure is already available (26). However, for the sake of
comparison with Ab12, we carried out the structure calculation of Ab16 under conditions similar to those at which
Ab12 structure was obtained. Such a study would provide
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Narayan et al.
A
Figure 6: (A) Amide region plots of titration experiments of Ab12 with AlCl3 at 298 K. Bar charts (Blue: free peptide and Red: metal
bound) showing the chemical shifts differences of (B) amide, and (C) Ca protons.
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Narayan et al.
Figure 10: ESIMS recording of Ab12 with aluminium metal. Free peptide peak is observed at 1424 Da, whereas aluminium metal-bound
peptide peak appear with highest intensity at 1448 Da. Peptide bound to two aluminium can also be identified as a low-intensity peat at
1472 Da.
Discussion
In literature, there are several reports on the interaction of
metals like zinc, copper, etc. (2327,37) with Ab peptides.
But, in spite of aluminium being implicated as a trigger for
the onset of Alzheimers disease (22), very limited NMR
studies involving aluminium have been reported. In a bioinformatics analysis carried out by us involving the residues of the Ab12 fragment in various metal-bound protein
structures in the protein data bank (45), we find that there
are very few aluminium-bound structures reported as
shown in Table S1. Of the reported structures, none are
NMR structures.
It is well known that one of the reasons for the plaque formation is the metal-induced aggregation of Amyloid peptide Ab42. It is also known that the C-terminus residues
being hydrophobic are generally found inside the membrane, and hence, the exposed region of 128 residues
Chem Biol Drug Des 2013; 82: 4859
(Ab28) is responsible for aggregation leading to plaque formation (6). Further the focus of several studies have been
to 116 residues as it was shown that His13, and His14
are implicated in metal binding (39).
Conformational changes in peptides and proteins induced
by aluminium (46) have been considered as neurotoxic
(47).and recent evidences have shown that the smaller
fragments of Ab are produced in presence of elastase
(48). Hence, the present work on fragments 112 (Ab12)
and 116 (Ab16) becomes significant to understand possible structural changes and the relevance, if any, of His13
and His14 in metal binding. The NMR structures of these
fragments presented here were found to be very similar,
but for the extension at the C-terminus as shown in
Figure 4, and Al3+ titrations with Ab16 (which contained
His13 and His14) showed no interactions with the histidine
residues. On the other hand, the study reveals additional
plausible metal interaction sites specific to aluminium,
namely Gly9, Tyr10 and Glu11.
Conclusions
Metal interactions with Ab peptides are strongly implicated
in fibril formation.
This study was carried out as there is limited information
available on aluminium interactions with Ab peptides.
Such study becomes important as aluminium entry into
brain along with zinc and other metals has been clearly
identified. Aluminium in food packing and cooking vessels is common sources for aluminium entry into the
human system. Here, we show that aluminium does bind
to Ab fragments, but not at the generally expected histidine residue site. The binding does not bring about any
major structural changes but causes small local perturbations. Studies also show that there are no major structural changes when the 16 residue fragment Ab16 is
reduced to 12 residues Ab12, where the two important
metal-binding histidine residues (His13 and His14) along
with the remaining two Gln15 and Lys16 are deleted. In
both Ab12 and Ab16, the same set of resonances is
disturbed implying the interactions with aluminium are
similar.
There are attempts to reverse the effect of metal interaction by use of metal chelators (49). Curcumin and Betaine
have also been tested for such reversal, wherein they
compete with the metal-binding sites (3135). Further
investigations are under way in this direction.
Acknowledgments
We thank Dr Anjali Ganjiwale for her help in generating
CYANA structures. Financial support from DST, government of India, is gratefully acknowledged.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
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