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

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

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

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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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.

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Supplemental Figure 3: Optimization of DSF assay in 384 well plate. Results are the average of experiments performed in triplicate. Error bars represent SD.

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Supplemental Figure 7: Hsp60 works with Hsp10 to refold denatured pMDH.

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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.

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