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13
1
00 mm phosphate buffer (pH 7.4). The buffer solution had been
The H and C signals of the peptides were assigned on the basis
1
1
1
13
1
13
pre-treated with Chelex 100 resin (Bio-Rad) to remove any metal
traces. The absorbance was registered over a 30 min time span.
The inhibition rates were calculated according to Equation (3):
of H- H COSY, H- C HSQC, and H- C HMBC experiments. It
should be noted that, even though the measurements were made
in D O, it was decided to use the notation pH.
2
EPR studies: X-band EPR spectra of frozen solutions were recorded
at 77 K on a Bruker ESP300E spectrometer. WINEPR 2.11 software
(Bruker) was used to process and simulate the spectra. Aqueous
Abs À Abs
blank
%
inhibition ¼ ð1 À
Þx100
ð3Þ
Abs
À Abs
freCu
blank
2
+
CuCl /peptide mixtures in ratios of 1:1.1 (5 mm Cu ) and 1:100
2
2
+
Electrospray ionization mass spectrometry: ESI mass spectra
were recorded on an LC/MSD-TOF spectrometer (Agilent Technolo-
gies) equipped with an electrospray ionization (ESI) source at the
Serveis Cientificotcnics of the Universitat de Barcelona. The spec-
tra were acquired in positive-ion mode at either 175 or 215 V. The
(2 mm Cu ) were used in 100 mm HEPES buffer (pH 7.4).
Competitive binding studies using GFP-Ab(1–42) fusion con-
structs
Production and purification of bacterial inclusion bodies (IBs): Escheri-
chia coli competent cells BL21 (DE3) were transformed with the
pET28a vector from Novagen (Merck KGaA, Darmstadt, Germany)
carrying the DNA sequence of the Ab42-GFP fusion protein. The
Ab protein produced by the bacteria contains an additional me-
thionine residue at its N terminus for the addition of the initiation
codon ATG in front of the gene; hence, the sequence of the result-
ing [Met + Ab(1–42)] peptide is
solutions were prepared in Milli-Q water, with [CuCl ]=0.5 mm,
peptide]=0.25 mm, and pH 7.4 (adjusted with NaOH).
2
[
Fluorescence spectroscopy: Fluorescence measurements were car-
ried out using a HORIBA Jobin–Yvon iHR320 spectrofluorimeter at
room temperature. The photomultiplier detector voltage was set
at 950 V and the instrument excitation and emission slits were
both set at 5 nm. In all cases, the pH was fixed at 7.4 with 100 mm
HEPES buffer, except in the competition studies, for which 10 mm
HEPES was employed instead.
Met - Asp - Ala - Glu - Phe - Arg - His - Asp - Ser - Gly - Tyr - Glu -
Val - His - His - Gln - Lys - Leu - Val - Phe - Phe - Ala - Glu - Asp -
Val - Gly - Ser - Asn - Lys - Gly - Ala - Ile - Ile - Gly - Leu - Met - Val -
Gly - Gly - Val - Val - Ile - Ala. Over-expression of the Ab42-GFP
Calculation of the conditional binding constants: The apparent affini-
II
c
[60]
ty constants of the Cu complexes with HWH and HK H were cal-
fusion constructs was carried out as described previously. Briefly,
culated by fitting the fluorescence titration data to Equation (1)
bacterial cultures (10 mL) were grown overnight at 378C and
250 rpm in Luria broth (LB) medium containing 50 mgmL kana-
[41,42]
À1
(
see above).
Since HEPES, the buffer used for the measure-
ments, can form a 1:1 complex with copper(II) and can thus act as
a weak competitor, its influence on the binding equilibrium had to
be taken into account. Therefore, the affinity constants at zero
mycin. For the protein over-expression, these cultures (10 mL)
were inoculated into LB (1 L) containing 50 mgmL of kanamycin.
À1
At an absorbance at 600 nm of 0.8, 1 mm isopropyl-b-d-1-thioga-
lactopyranoside (IPTG) was added to induce recombinant protein
expression. After 4 h, the bacterial cells were harvested by centrifu-
gation, and the obtained pellets were re-suspended in lysis buffer
containing 100 mm NaCl, 1 mm EDTA, and 50 mm Tris at pH 8 to
purify the intracellular inclusion bodies (IBs), as described previous-
buffer concentration, that is, the conditional binding constants
cond
(K
), were determined. For very large quantities of HEPES with re-
spect to the other components in the solution, Equation (2) (see
[43]
above) can be applied.
Generation of hydroxyl radicals; formation of 7-hydroxy-coumarin-
carboxylic acid (7-OHCCA): Single-point fluorescence measurements
were performed at room temperature in phosphate buffer
[19]
ly. The IBs were purified by a differential centrifugation/deter-
[61,62]
gent washing procedure.
In brief, the protease inhibitor PMSF
and lysozyme were added so as to give final concentrations of
(
100 mm, pH 7.4). The buffer solution had been pre-treated with
À1
1
5 mm and 300 mgmL , respectively. The samples were then incu-
Chelex 100 resin (Bio-Rad) to remove any metal traces. The samples
were excited at 385 nm and the emission at 500 nm was registered
over a period of 30 min, under aerobic conditions. A 5 mm stock
solution of 3-coumarin-carboxylic-acid (3-CCA) was prepared in
bated at 378C for 30 min. Subsequently, 1% NP-40 was added and
the mixture was incubated at 48C for 50 min under gentle agita-
tion. In order to remove nucleic acids, the cells were treated with
À1
[53]
15 mgmL DNase and RNase at 378C for 30 min. Finally, the IBs
phosphate buffer as described previously. An ascorbate solution
was freshly prepared in phosphate buffer immediately prior to
each measurement. The final concentrations used for the assays
were collected by centrifugation at 12,000 g for 10 min and
washed with lysis buffer containing 0.5% Triton X-100. In a last
[62]
step, the samples were washed three times with PBS.
were 1 mm CuCl , 1.1 mm peptide, 300 mm ascorbic acid, and 1 mm
2
3
-CCA. The inhibition rates were calculated according to Equa-
In vitro refolding assay of Ab42-GFP inclusion bodies: Purified IBs
tion (4):
were concentrated by centrifugation to an absorptivity of 100 at
3
60 nm. Then, 10 mL of IBs was centrifuged and the pellets were
I À I
re-suspended in 10 mL of 8m guanidine (Gdn·HCl) and incubated
at room temperature for 4 h under mild agitation and sonication.
blank
%
inhibition ¼ ðI À
Þx100
ð4Þ
I
À Iblank
freeCu
For the refolding process, 10 mL of denatured IBs was dissolved in
990 mL of refolding buffer. These buffers were prepared from PBS
that had been pre-treated with Chelex 100 from Sigma–Aldrich (St.
Louis, MO, USA). When required, 10 mL of refolding buffer was re-
NMR studies: 1D and 2D NMR experiments were performed on
a Bruker Avance III 400 MHz spectrometer, equipped with a 5 mm
1
19
cryoprobe (Prodigy) broadband (CPPBBO BB- H/ F/D) with gradi-
ents in Z, at the Centres Científics i Tecnològics of the Universitat
de Barcelona (CCiTUB). Solvent suppression for the 1D experiments
was achieved using a PRESAT pulse sequence. The NMR data were
analyzed using MestReNova 9.1.0. Solutions of 10 mm peptide in
placed by 10 mL of 1 mm CuCl so as to give a final metal concen-
2
tration of 10 mm. In the same manner, 10 mL aliquots of HWH,
C
HK H, or HAH from 1 mm stock solutions were added to the
sample. The samples were then incubated at room temperature
for 16 h (overnight) under mild agitation. The GFP fluorescence of
the solutions containing refolded IBs was measured with an
Aminco Bowman Series 2 luminescence spectrophotometer
(Aminco-Bowman AB2, SLM Aminco, Rochester, NY, USA), using ex-
citation and emission wavelengths of 488 nm and 512 nm, respec-
D O were adjusted to pH 7.4, and measured with an insert contain-
2
À1
ing a 5 mgmL solution of [D ]-3-(trimethylsilyl)propanoic acid
TSP) as an internal reference. In the presence of 0.01 equiv of Cu
4
II
(
ions, the intensity of the peptide NMR signals decreased by about
0%.
6
Chem. Eur. J. 2016, 22, 7268 – 7280
7278
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim