close

Вход

Log in using OpenID

embedDownload
Supporting Information
Helix Stabilized, Thermostable, and Protease-Resistant Self-Assembled
Peptide Nanostructures as Potential Inhibitors of Protein-Protein
Interactions
Woo-jin Jeong,†,‡, Myeong Sup Lee,§, and Yong-beom Lim*,†,‡
†
Translational Research Center for Protein Function Control, ‡Department of Materials
Science & Engineering, and §Department of Biochemistry, Yonsei University, Seoul 120749, Korea
‖
These authors contributed equally to this work.
*E-mail: yblim@yonsei.ac.kr
(1) Monomeric p5317-28 peptide
H-GETFSDLWKLLPEC-NH2
(2) Fluorescein-p5317-28
FAM-GETFSDLWKLLPEC-NH2
(3) A model peptide with a linker equivalent to the length of ~6 amino acids
cyclo[-(Sak)-(Ebes)-WKWEWYWKWEW-(Ebes)-SAEAAAKEAAAKAC-]
O
Sak
S
Ebes
N
H
O
O
O
H
N
O
(4) A model peptide with a linker equivalent to the length of ~5 amino acids
cyclo[-(Sak)-GSGWKWEWYWKWEW-(Ebes)-AEAAAKEAAAKAC-]
(5) A model peptide with a linker equivalent to the length of ~4 amino acids
cyclo[-(Sak)-SGWKWEWYWKWEWSGSGAEAAAKEAAAKAC-]
(6) p53 g-peptide
cyclo[-(Sak)-SGWKWEWYWKWEWSGSGETFSDLWKLLPEC-]
(7) Oligo(EG)-p53 g-peptide
cyclo[-(Sak)-SGWKWEWYWKWEW-{ε-K[α-(Ebes)3]}-SGETFSDLWKLLPEC-]
Figure S1. Sequence of peptides.
H2N
H
N
O
O
O
O H
N
OH
O
H
N
H
N
O
H
N
O
NH2
O
O HN
NH
O
NH
O HN
O
HO
NH
O HN
O
HO
NH2
NH
O HN
O
S
HN
O
O
NH
O
HN
O
O
O
O
NH
HO
O H
N
HO
O
NH O
HN
O
HO
O
HN
O
N
H
O
NH
N
H
O
NH
H
N
O
NH
O
NH
NH2
O
N
O HN
OH
NH
O
N
H O
NH
HO
O
O
H
N
OHO HN
NH O
H
N
N
H
O
O
O H
N
O
NH
NH O
HN
H2N
HN
O
O
HN
HN
O
O
O
OH
NH
HN
O
NH NH2
O
O
H2N
HN
O
O
OH
S
HO
(3) A model peptide with a linker equivalent to the length of ~6 amino acids
O
NH
NH
O
NH
O
HO
OH
O
HN
HN
NH
O H
N
O
H2N
O
NH
OH
NH O
O
O
O
O H
N
H
N
O
H
N
O
HO
NH
O HN
O
HN
N
H
H
N
O
NH
HN
N
H
O
NH
O
NH
NH O
N
H
O
O HN
HO
NH2
NH O
O
O
NH2
N
H
N
H O
O
O
NH
HO
O
NH
O
O HN
OH
NH
O
NH NH2
HN
O
NH
H2N
(6) p53 g-peptide
O
O
S
NH
O
NH
O
O
NH
O
OH
O
HN
HN
NH
O
O
HO
O H
N
HO
NH
H2N
HN
O
NH
O
O H
N
H
N
O
H
N
O
NH
O
HN
NH O
O
O
NH
OHO HN
O
HN
O
HO
O
NH O
HN
NH
N
H
O
O
NH
N
H
N
H
N
H O
O
O
NH
HO
N
O HN
OH
O
O
O
OH
NH
O
NH
H2 N
NH2
NH
NH
O
O
O
NH NH2
HN
OH
HN
O
O
(4) A model peptide with a linker equivalent to the length of ~5 amino acids
HN
O
O
O
O
S
HO HN
NH
OO
O
HN
NH
O
O
OH
O
NH
O
HN
HN
NH
H2N
O
O
OH
O
O
O H
N
H
N
O
H
N
H
N
NH
O
O
HO
O
O
NH O
HN
O
NH
O
NH
O HN
HN
O
NH2
NH2
NH
O HN
HO
NH O
O
O
O
NH
N
H
O
NH2
O
N
H
O
N
H O
N
H
O
NHO
NH
O
OH
OH
NH
O
HN
O
NH NH2
HN
O
O
S
HO
O
NH
NH
O
NH
O
HO
OH
O
HN
HN
NH
O H
N
O
NH
NH O
O
N
H
HN
HO
O
O
NH2
NH O
HN
O
NH
H2N
NH O
N
H
O
N
H
O
N
H O
N
H
NH
O
NH
HO
O
NH
OH
(5) A model peptide with a linker equivalent to the length of ~4 amino acids
Figure S2. Chemical structures of cyclic peptides.
O NH
NH
HN
O HN
NH
O
O
(7) Oligo(EG)-p53 g-peptide
NH
NH2
Figure S3. MALDI-TOF MS spectra. a, Monomeric p5317-28 peptide. b, Fluorescein-p5317-28.
c, A model peptide with a linker equivalent to the length of ~6 amino acids. d, A model
peptide with a linker equivalent to the length of ~5 amino acids. e, A model peptide with a
linker equivalent to the length of ~4 amino acids. f, p53 g-peptide. g, Oligo(EG)-p53 gpeptide.
Figure S4. His6-tagged MDM2 protein expression and purification of tag-free MDM2. a,
Thrombin cleavage reaction to remove His6-tag from the expressed protein. b, Purification of
MDM2 protein from the thrombin cleavage reaction mixture by gel filtration chromatography.
c, SDS-PAGE of purified MDM2 protein. d, MALDI-TOF MS spectrum of purified MDM2
protein.
Figure S5. DLS autocorrelation function for p53 αSSPN.
EXPERIMENTAL SECTION
Peptide synthesis and cyclization procedures
Peptides were synthesized on a Rink Amide MBHA resin LL (Novabiochem) using
standard Fmoc protocols on a TributeTM peptide synthesizer (Protein Technologies, Inc).
Standard amino acid protecting groups were employed except Cys(Mmt) and Lys(Dde), in
which an acid-labile methoxytrityl (Mmt) and N‐[1‐(4,4‐dimethyl‐2,6‐dioxocyclohex‐1‐
ylidene)ethyl] (Dde) groups were used, respectively. The oligoethylene glycol-based
linker, N-(Fmoc-8-amino-3,6-dioxaoctyl)succinamic acid (Fmoc-PEG2-Suc-OH or
Fmoc-Ebes-OH), was purchased from Anaspec.
For cyclization, the peptide-attached resin (20 µmol of N-terminal amine groups)
was swollen in N-methyl-2-pyrrolidone (NMP) for 30 min. Then, bromoacetic acid
was first coupled to the N-terminal part of the resin-bound peptide. Before addition to
the resin, a mixture of bromoacetic acid (28 mg, 200 µmol) and N,N′diisopropylcarbodiimide (15.5 µL, 100 µmol) in NMP was incubated for 10 min for
carboxyl activation. Following addition of the mixture to the resin, the reaction was
continued for 1 h with shaking at room temperature, in a 6 mL polypropylene tube
with a frit (Restek). The resin was then washed successively with NMP and
dichloromethane (DCM). For orthogonal deprotection of the Mmt group from the
cysteine, the resin was treated with 1% trifluoroacetic acid (TFA) in DCM several
times (1 min × ~7). Intramolecular cyclization reaction was performed in 3 mL of 1%
diisopropylethylamine (DIPEA) in NMP overnight with shaking at room temperature.
The resin was then successively washed with NMP and DMF.
Deprotection of Dde from Lys(Dde) was performed in 2% hydrazine in DMF.
Then coupling of the solubility enhancer, Fmoc-Ebes-OH, was followed using
standard Fmoc protocols.
For cleavage and final deprotection, the resin was treated with cleavage cocktail
(TFA:1,2-ethanedithiol:thioanisole; 95:2.5:2.5) for 3 h, and was triturated with tert-
butyl methyl ether (TBME). The peptides were purified by reverse-phase HPLC
(water−acetonitrile with 0.1% TFA). The molecular weight was confirmed by MALDITOF mass spectrometry. The purity of the peptides was >95% as determined by
analytical HPLC. The peptide concentration was determined spectrophotometrically
in 8 M urea using a molar extinction coefficient of tryptophan (5,500 M-1cm-1) at 280
nm.
Circular dichroism (CD) spectroscopy
CD spectra were measured using a ChirascanTM circular dichroism spectrometer
equipped with a Peltier temperature controller (Applied Photophysics). The spectra
were recorded from 260 to 190nm using a 2 mm path-length cuvette. Scans were
repeated five times and averaged. Molar ellipticity was calculated per amino acid
residue.
Dynamic light scattering
DLS experiments were performed at room temperature using an ALV/CGS-3
compact goniometer system equipped with a He-Ne laser operating at 632.8 nm.
The detector optics employed optical fibers coupled to an ALV/SO-SIPD/DUAL
detection unit, which employed an EMI PM-28B power supply and an ALV/PM-PD
preamplifier/discriminator. The signal analyzer was an ALV-5000/E/WIN multiple-tau
digital correlator with 288 exponentially spaced channels. The scattering angle was
90º. The size distribution was determined using a constrained-regularization method.
Fourier transform infrared spectroscopy (FT-IR)
For FT-IR measurement, 100 μL of the sample (50 μM in water) was cast from
the solution onto ZnSe window. Three thousand scans were acquired on a Bruker
VERTEX 70 FT-IR spectrometer.
Atomic force microscopy (AFM)
For AFM, typically 2 μL of the sample in water was deposited onto a freshly cleaved
mica surface and dried in air for overnight. The images were obtained in tapping mode with a
Nanoscope IV instrument (Digital Instruments). AFM scans were taken at setpoint of 0.8-1 V
and scanning speed was 1-2 Hz.
Transmission electron microscopy (TEM)
Two µL samples were placed onto a carbon-coated copper grid and dried
completely. Then 2 μL of 2 % (w/v) uranyl acetate solution was added for 1 min and
excess solution was wicked off by filter paper. Sample concentrations were typically
5-20 μM. The specimens were observed using a JEOL-JEM 2010 instrument
operating at 120 kV. The data were analyzed using DigitalMicrographTM software.
Protein expression and purification
The six His (His6)-tagged, recombinant human MDM2 (hDM2) protein (N-terminal
domain spanning amino acids 17-125) was overexpressed in E. coli. The plasmid pET28ahMDM2(17-125) [Kanamycin-resistance (KanR), kindly provided by Dr. Gregory Verdine,
Harvard.] expressing the truncated human hDM2 (a.a. 17-125) with N-terminal six His
(His6)-tag, together with thrombin cleavage site was introduced into E. coli strain BL-21Codon Plus (DE3)-RIPL [Chloramphenicol-resistance (CmR), Agilent Technologies] using
standard chemical transformation method. The transformed single colony was cultured in LB
media with the drugs Cm (25 µg/mL) and Kan (20 µg/mL) at 30 °C until optical density (OD)
at 600 nm reaches ~0.5, then treated with isopropyl β-D-thiogalactoside (IPTG; 0.1 mM) at
16 °C for 18 h to induce the recombinant protein. The E. coli was collected by spinning the
culture at 6000 rpm (Sorvall) for 10 min at 4 °C.
All the purification steps were performed at 4 °C, unless specified otherwise. The E. coli
cell pellet was resuspended in the lysis buffer [20 mM Tris⋅HCl pH 8.0, 500 mM NaCl, 10%
glycerol, 20 mM imidazole, 0.1% (v/v) NP-40, 5 mM β-mercaptoethanol] with protease
inhibitor cocktail (1 mM PMSF, 10 µg/ml aprotinin, 5 µg/ml pepstatin and 5 µg/ml leupeptin)
and treated with lysozyme (1 mg/ml) for 45 min to lyse the bacterial cell wall. Then the
resuspended cells were sonicated to break the cells and the viscous genomic DNA until the
cell lysate is not viscous any more. After removing cell debris by centrifugation at 14, 000 ×
g for 10 min, the recombinant protein from the cell lysate was purified in the lysis buffer
using Ni-NTA agarose beads following the standard purification protocol provided by the
vendor (Qiagen), except the elution which was performed in the lysis buffer with 250 mM
imidazole.
For the removal of His6-tag from the recombinant human MDM2 protein, 0.5 U of
thrombin was mixed with 20 µg of the hDM2 protein in thrombin cleavage buffer (50 mM
Tris⋅HCl pH 7.5, 150 mM NaCl, 2.5 mM CaCl2). The sample was incubated at 4 °C overnight.
Progress of the cleavage reaction was monitored by SDS-PAGE. The sample was subjected to
gel filtration chromatography using a Superdex 75 column at 4 °C. Elution buffer was 50 mM
Tris pH 8.0, 140 mM NaCl. The sample purity and identity were confirmed by SDS-PAGE
and MALDI-TOF mass spectrometry (Supplementary Fig. 4). The concentration of purified
MDM2 protein was determined by Micro BCA protein assay (Pierce ).
Interaction and competition assay
The protein-protein interaction assays were performed using a fluorescence polarization
(FP) assay in a buffer containing 50 mM Tris pH 8.0, 140 mM NaCl at room temperature.
Fluorescence anisotropy measurements were done in a 384-well plate using a Victor X5
multilabel plate reader (Perkin Elmer).
The dissociation constant (Kd) was calculated by fitting experimental data to the
following explicit equation, which is based on single-site binding model1-3.
0 i 0 MDM2 ligand d MDM2 ligand d2 4MDM2ligand
2ligand
A = measured fluorescence anisotropy
Ai = fluorescence anisotropy of ligand in the presence of an infinite concentration of
MDM2
A0 = fluorescence anisotropy of in the absence of MDM2
[MDM2] = concentration of MDM2 protein
[ligand] = concentration of ligand, Fluorescein-p5317-28
[MDM2] increased progressively, while [ligand] held constant. [Fluorescein-p5317-28] was
2.4 nM.
Competition data were fitted to a single-site binding model with variable Hill slope.
0 i 0/1 10
50!"#$%&'")*+,
For FP competition assay, FAM-p5317-28/MDM2 complexes (2.4 nM/3000 nM; the
MDM2 concentration was more than 3-fold greater than Kd) was titrated with an unlabeled
competitor. The data were analyzed using GraphPad Prism and SigmaPlot software.
Protease resistance experiment
For the peptide digestion4, Chymotrypsin was added to the samples (final
peptide:chymotrypsin mass ratio was 5:1) and incubated at 37 °C. Aliquots were taken from
the samples at 0, 1, 5, 15, 30, 60, and 120 min, and were immediately treated with TFA to a
final concentration of 0.1 % (v/v) in order to quench the digestion reaction. Before analysis,
self-assembled peptide nanostructures were disassembled by treating with 1,1,1,3,3,3hexafluoro-2-propanol (HFIP), a strong β-breaker5, 6, to a final concentration of 50% (v/v) for
30 min. The samples were then monitored chromatographically using a reversed phase HPLC
(water/acetonitrile with 0.1% TFA) and the recovery was calculated by integration of
the 280 nm signal. The molecular weight was confirmed by MALDI-TOF mass
spectrometry.
1.
Bernal, F.; Tyler, A.F.; Korsmeyer, S. J.; Walensky, L. D.; Verdine, G. L. J. Am. Chem. Soc. 2007, 129,
2456-2457.
2.
Cho, J. H.; Rando, R. R. Biochemistry 1999, 38, 8548-8554.
3.
Kirk, S. R.; Luedtke, N. W.; Tor, Y. J. Am. Chem. Soc. 2000, 122, 980-981.
4.
Getz, J. A.; Rice, J. J.; Daugherty, P. S. ACS Chem. Biol. 2011, 6, 837-844.
5.
Hirota, N.; Mizuno, K.; Goto, Y. Protein Sci. 1997, 6, 416-421.
6.
Wood, S. J.; Maleeff, B.; Hart, T.; Wetzel, R. J. Mol. Biol. 1996, 256, 870-877.
7.
Hansen, M. B.; Nielsen, S. E.; Berg, K. J. Immunol. Methods 1989, 119, 203-210.
1/--pages
Пожаловаться на содержимое документа