shao orgbiochem supplemental text · and 52 µl of 0.05m atp. after a 90 min incubation at 30 °c,...
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![Page 1: Shao OrgBioChem Supplemental text · and 52 µL of 0.05M ATP. After a 90 min incubation at 30 °C, the protein mixture was purified by SEC200 column pH(Running buffer: 50 mM Tris-HC1,](https://reader035.vdocuments.net/reader035/viewer/2022070919/5fb8de826affe737fd628f79/html5/thumbnails/1.jpg)
Supplemental Information Differential Scanning Fluorimetry (DSF) Screen to Identify Inhibitors of
Hsp60 Protein-Protein Interactions
Hao Shao, Keely Oltion, Taia Wu and Jason E. Gestwicki*
Experimental Protein expression and purification
The purification of Hsp60 was modified from a previously reported method.1 Briefly,
human Hsp60 was expressed in E. coli BL21 (DE3) cells. The BL21 cells in 1L of terrific
broth (TB) were grown at 37 °C with shaking until the OD600 reached 0.6. Then, the
cultures were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG; final
concentration 300 µM) and grown at 37 °C for 4 hours. For protein purification, cell pellets
were re-suspended in His-binding buffer (50 mM TRIS, 10 mM Imidazole, 500 mM NaCl,
pH 8) supplemented with protease inhibitors. Cells were then lysed by sonication, pelleted
by centrifugation, and the supernatant was incubated with Ni-NTA His-Bind Resin for 1
hour at 4 °C (Novagen, Germany). The resin was washed with His-binding buffer, followed
by His-washing buffer (50 mM TRIS, 30 mM Imidazole, 300 mM NaCl, pH 8). The protein
was then removed from the resin using His-elution buffer (50 mM TRIS, 300 mM
Imidazole, 300 mM NaCl, pH 8). The N-terminal His-tags were removed by incubating the
protein and TEV protease with 1 mM DTT for 4 hours at room temperature, followed by
dialysis in 4 L of SEC buffer (50 mM Tris-HC1, 0.3 M NaC1, 10 mM MgC12, pH 7.70)
overnight. The second day, the uncleaved protein was removed by a reverse Ni-NTA
purification and concentrated. To assemble Hsp60 oligomers, the following were mixed:
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2020
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573 µL of Hsp60 (purified as monomer), 13 µL of 1M KC1, 13 µL of 1M magnesium acetate,
and 52 µL of 0.05M ATP. After a 90 min incubation at 30 °C, the protein mixture was purified
by SEC200 column (Running buffer: 50 mM Tris-HC1, 0.3 M NaC1, 10 mM MgC12, pH 7.7).
The pooled oligomers are concentrated to 15-20 mg/mL, aliquoted, flash- frozen, and stored at
-80 °C.
Differential Scanning Fluorimetry Assay DSF experiments were performed in a BioRad CFX connect or Roche LC480 Light Cycler II
qPCR machine in the FRET channel. Solutions of protein (assay buffer: 50 mM Tris, 20 mM
KCl, 10 mM MgCl2, 200 mM NaCl, PH 7.4), Sypro Orange (Thermo Fisher Scientific, S6650)
and tested compounds or DMSO were added to the wells of a 96-well PCR plate (USA
Scientific, 1402-9590) or 384-well microtiter plate (Axygen, PCR-284- LC480WNFBC). The
plates were sealed with transparent films (EXCEL Scientific, TS- RT2-100) and heated in the
qPCR machine from 25 to 95 °C in increments of 1.0 °C. The 96 well-plate assay data was
analyzed using DSFworld (Wu et al submitted) using the first derivative and Boltzmann models.
The 384-well assay data was analyzed using Roche LC480 Light Cycler II software. Technical
note: In our experiments, we have noted that signal intensity is highly dependent on the plastic
used in microtiter plates, even showing differences between “lots”. Using different qPCR
machines also cause signal intensity variability. So, one should not read into that value between
experiments.
Malachite Green ATPase Assay The ATPase activity assay was adopted from a previous method, with modifications.2 The
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malachite green reagent was prepared from stock solutions three hours before use (malachite
green (0.081% w/v), polyvinyl alcohol (2.3% w/v), ammonium heptamolybdate tetrahydrate
(5.7% w/v in 6 M HCl) and water (ratio of 2:1:1:2). To each well of a 96-well plate, 1 µL
compounds or DMSO were added to 19 µL Hsp60/Hsp10 solution (2.63 µM and 5.26 µM
respectively) in assay buffer (100 mM Tris, 20 mM KCl, 10 mM MgCl2, 0.017% Triton, pH
7.4). To this solution, 5 µL of 5 mM ATP was added to initiate the reaction, such that the final
reaction volume was 25 µL and the conditions were 2.0 µM Hsp60, 4 µM Hsp10, 4% DMSO,
0.01% Triton X-100, and 1 mM ATP. After 3 h incubation at 37 °C, 80 µL of malachite green
reagent was added into each well, followed by 10 µL 32% sodium citrate to limit chemical
hydrolysis of ATP. The plate was incubated at 37 °C for 15 min before measuring OD620 on a
SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA).
Native Gel Assay A solution of Hsp60 (5 µM) was treated with compounds at the indicated concentrations for 30
mins. Then sample buffer (5X: 0.05% Ponceau Red, 50% Glycerol, 250 mM 6- aminohexanoic
acid, 50 mM Bis-Tris, pH 7.0) was added. An aliquot of 15 µL and molecular weight marker
(NativeMark Unstained Protein Standard-Invitrogen) were loaded on precast 4–16% Novex
NativePAGE Bis-Tris gels. The electrophoresis was performed at 150 V constant voltage for 4
hours at room temperature with 50 mM Bis Tris (pH 7.0) as anode buffer and 15 mM Bis Tris
and 50 mM Tricine (PH 7.0) as Cathode buffer. Gel was stained by Coomassie G-250 and
scanned using ChemiDoc Touch Imager.
Refolding of Denatured MDH
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The assay was adopted from a previously published protocol with modification.3 Briefly, malate
dehydrogenase (MDH, Sigma-10127256001) was denatured by incubating 94 µM MDH with
one equivalent guanidine buffer (7 M guanidine•HCl, 200 mM Tris, pH 7.4) for 1 h at room
temperature. Then, the denatured MDH (120 nM) was added to Hsp60 (3.33 µM) in refolding
buffer (100 mM Tris pH 7.4, 20 mM KCl, 10 mM MgCl2, and 1 mM DTT). After ~10 mins at
room temperature, Hsp10 (6.67 µM) was added. Then, 30 µL of this solution was dispensed
into clear 384-well plates (CORNING, 3702), followed by addition of 1 µL compound A10 or
DMSO. Refolding was initiated by addition of 20 µL of 2.5 mM ATP solution (concentrations
during refolding cycle: 2 µM Hsp60, 4 µM Hsp10, 72 nM denatured MDH, 1 mM ATP). Finally,
after incubating at 37 °C for an hour, the reaction was quenched by adding 10 µL of 500 mM
EDTA solution. To determine the amount of MDH that was re-folded, reaction progress was
monitored by measuring NADH absorbance at 340 nM for 60 minutes in assay buffer (20 mM
sodium mesoxalate monohydrate and 2.4 mM NADH in reaction buffer, Sigma-Aldrich). A340
measurements were recorded at T0 and then the time point at which 90% of substrate was
consumed was reported. The IC50 values were calculated using GraphPad Prism.
1. Viitanen PV, Lorimer G, Bergmeier W, Weiss C, Kessel M, Goloubinoff P. Purification of mammalian mitochondrial chaperonin 60 through in vitro reconstitution of active oligomers. Method Enzymol. 1998;290: 203-217. 2. Chang L, Bertelsen EB, Wisen S, Larsen EM, Zuiderweg ERP, Gestwicki JE. High- throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK. Anal Biochem. 2008;372(2): 167-176. 3. Johnson SM, Sharif O, Mak PA, et al. A biochemical screen for GroEL/GroES inhibitors. Bioorganic & medicinal chemistry letters. 2014;24(3): 786-789.
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mtH
sp60
Run
3001
:10_
UV
mtH
sp60
Run
3001
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Con
d m
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60 R
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0_C
ond%
mtH
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Run
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Con
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Run
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Frac
tions
mtH
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Run
3001
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Inje
ct m
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01:1
0_Lo
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-100
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200
300
400
mAU
50
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ml
12
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56
78
910
1112
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1516
1718
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2122
2324
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aste
Olig
omer
Mon
omer
Supp
lem
enta
l Fig
ure
1: S
EC p
urifi
catio
n tra
ces
for H
sp60
olig
omer
and
mon
omer
.
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Supplemental Figure 2: Optimization of DSF assay in 96 well plate. Investigate (a) SYPRO Orange concentration, (b) DMSO concentration, (c) sample volume, and (d) Hsp60 oligomer concentration’s effect on Tm. Results are the average of experiments performed in triplicate. Error bars represent SD.
40 60 800
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Fluo
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ence
50x10x
2.5x1x
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Temperature (°C)
Fluo
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10%5%1%0%
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ence
20 µL15 µL10 µL5 µL
25 µL
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Temperature (°C)
Fluo
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ence
5 µM2.5 µM2 µM1.5 µM1.25 µM1 µM.75 µM.25 µM0 µM
(a) SYPRO orange concentration
(b) DMSO concentration
(c) Sample Volume
Temperature (°C)
Temperature (°C)(d) Hsp60 oligomer concentration
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40 60 80 1000
5
10
15
(c) 2.5 uM Hsp60 oligomer
Fluo
resc
ence
(a) 4 uM Hsp60 oligomer
10 µL15 µL20 µL25 µL
10 µL15 µL20 µL25 µL
40 60 80 1000
5
10
15(b) 3 uM Hsp60 oligomer
Fluo
resc
ence
10 µL15 µL20 µL25 µL
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(d) 2 uM Hsp60 oligomer
Fluo
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25 µL20 µL15 µL10 µL
40 60 80 100
-1.0
-0.5
0.0
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dF/d
T40 60 80 100
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T
Supplemental Figure 3: Optimization of DSF assay in 384 well plate. Results are the average of experiments performed in triplicate. Error bars represent SD.
40 60 80 1000
5
10
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20
Temperature °C
Fluo
resc
ence
Temperature °C
Temperature °C
Temperature °C
Temperature °C
Temperature °C
Temperature °C
Temperature °C
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Current Chromatogram(s)
min0.5 1 1.5 2 2.5 3 3.5
50000100000150000200000250000300000350000400000450000
MSD1 TIC, MS File (D:\DATA\2020-03-11\IND 2020-03-11 11-30-55\A10.D) ES-API, Pos, Scan, Frag: 70
0.18
5
0.36
90.
426
0.50
50.
577
0.61
0
0.74
20.
770
0.83
30.
891
0.96
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281
1.39
8
1.58
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1.78
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1.92
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2.40
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2.59
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670
2.78
82.
835
2.98
23.
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3.06
73.
157
3.25
83.
304
3.42
5
3.55
23.
662
3.84
63.
908
min0.5 1 1.5 2 2.5 3 3.5
mAU
0
500
1000
1500
2000
MWD1 A, Sig=215,4 Ref=off (D:\DATA\2020-03-11\IND 2020-03-11 11-30-55\A10.D)
2.36
6
min0.5 1 1.5 2 2.5 3 3.5
mAU
0
500
1000
1500
2000
2500
MWD1 B, Sig=254,4 Ref=off (D:\DATA\2020-03-11\IND 2020-03-11 11-30-55\A10.D)
2.36
7
min0.5 1 1.5 2 2.5 3 3.5
mAU
0250500750
1000125015001750
MWD1 E, Sig=280,4 Ref=off (D:\DATA\2020-03-11\IND 2020-03-11 11-30-55\A10.D)
2.36
7
MS Spectrum
m/z340 360 380 400
0
20
40
60
80
100
*MSD1 SPC, time=2.418 of D:\DATA\2020-03-11\IND 2020-03-11 11-30-55\A10.D ES-API, Pos, Scan, Frag: 70
Max: 103528
356.
035
7.0
358.
035
9.0
378.
037
9.1
379.
938
0.9
Supplemental Figure 4: LC-MS spectrum of compound A10
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10 20 30 40-20
0
20
40
60
80
100
Time (min)
Rel
ativ
e pM
DH
act
ivity Hsp60 + Hsp10
Hsp60 alone
Supplemental Figure 7: Hsp60 works with Hsp10 to refold denatured pMDH.
40 60 80 1000
5000
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20000
Temperature (°C)
Fluo
resc
ence
20 µM10 µM5 µM1 µM0 µM
Supplemental Figure 5: The melting curves of Hsp60 oligomer treated with compound A10 at indicated concentrations. Results are the average of experiments performed in triplicate. Error bars represent SD.
EC50 = 5.0 µM
Supplemental Figure 6: A10 increased monomer population in a dose-dependent manner.
-0.5 0.0 0.5 1.0 1.5 2.00
50
100
Log[A10, µM]
Perc
enta
ge o
f mon
omer