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Electronic Supporting Information

The Other Side of the Corona: Nanoparticles Inhibit the Protease Taspase1 in a Size-Dependent Manner

Johannes van den Boom*, Astrid Hensel, Franziska Trusch, Anja Matena, Svenja Siemer, Désirée Guel, Dominic Docter, Alexander Höing, Peter Bayer, Roland H. Stauber, Shirley K. Knauer*

Electronic Supplementary Material (ESI) for Nanoscale.This journal is © The Royal Society of Chemistry 2020

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Table of Contents

1. Experimental Section (including References) S3

Nanoparticle characterization S3

Expression plasmids S3

Protein expression and purification S4

Fluorescent protein labeling S4

MALDI-TOF mass spectrometry S4

2. Electronic Supporting Figures S5

Fig. S1. Taspase1 is a type II asparaginase. S5

Fig. S2. Taspase1 protein expression and purification. S6

Fig. S3. Taspase1 secondary structure and stability. S8

Fig. S4. Taspase1 molecular weight, multimerization and autocatalytic processing. S9

Fig. S5. Taspase1 proteolytic activity. S10

Fig. S6. Structural models of Taspase1. S11

3. Electronic Supporting Tables S12

Table S1. Nanoparticle characterization. S12

Table S2. Parameters used for anisotropy measurements. S12

Table S3. Parameters used for kinetic measurements with the fluorogenic assay. S12

Table S4. Parameters used for recording of far-UV CD spectra. S13

Table S5. Parameters used for recording of fluorescence melting curves. S13

Table S6. Catalytic parameters of Taspase1 target sequences at 37 °C. S13

Table S7. Channel settings used for fluorescence microscopy. S14

4. References S14

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1. Experimental Section

Nanoparticle characterization

Nanoparticles were characterized concerning the shape, size and zeta potential in dry state or

solution. Transmission electron microscopy imaging was performed on carbon coated copper

grids as described ).1 Hydrodynamic sizes and the zeta potential were measured with a

Malvern Zetasizer NanoZS as described before at 25 °C and 0.6 mg/mL concentration ).1

Expression plasmids

The modified pET-22b vector containing wild-type Taspase1 cDNA with a C-terminal His-tag

was described previously.2 The bicistronic expression construct referred to as active Taspase1

is based on the construct described by Khan et al.3 It comprises the a-subunit with an N-

terminal hexa-histidine tag, followed by a stop codon and a second ribosomal binding site for

the b-subunit. The unstructured loop at the C-terminus of the a-subunit was shortened by 27

amino acids, ending at Ala 206, and codon usage was optimized for expression in E. coli using

OPTIMIZER.4 The gene was synthesized and cloned into a modified pET-41b vector (GeneArt).

The inactive mutant was generated from the described wild-type construct of human

Taspase1 cDNA in a pET-22b vector via insertion of two mutations in the active site (D233A

and T234A) using the QuikChange kit (Agilent) to prevent autocatalytic activation of the

proenzyme and hence reduce catalytic activity. The pRARE2 plasmid (Merck) containing the

tRNA for codons rarely used by E. coli has been described previously.5 Eukaryotic expression

constructs encoding human wild-type and inactive Taspase1 fusions with the red

autofluorescent protein mCherry (mCh) have been described.2, 6 The Taspase1 biosensor

(BioTasp, Figure 4b), serving as fluorescent reporter substrate encodes a fusion protein

composed of the SV40 large T-antigen nuclear localization signal (NLS), GST, GFP, and the

preferred second Taspase1 cleavage site derived from the MLL protein (CS2: aa

2713KISQLDGVDD2722) preceding a Myc-epitope-tagged nuclear export signal (NES) from the

HIV-1 Rev protein has been described.2, 6

Protein expression and purification

pET-22b containing wild-type Taspase1 was expressed as described.2

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Fluorescent protein labeling

Wild-type and inactive Taspase1 and the control enzyme parvalbumin were labeled with the

cysteine reactive dye Atto488-maleimide. The buffer was changed for labeling to 50 mM

NaH2PO4, 450 mM NaCl, pH 7.4 by repeated dilution and concentration in a 30 kDa cut-off

Centricon. A 2.5-fold molar excess of Atto488 (dissolved in DMSO) was added and the solution

was incubated for 60 min at room temperature (RT) in the dark. Unbound dye was removed

with the help of a PD-10 column and the conjugate was concentrated in a Centricon. To

determine the protein concentration (c) and degree of labeling (DOL), the absorbance at

280 nm and 601 nm was measured using a NanoDrop ND 1000. After labeling was completed,

the buffer was changed to the previous storage buffer (50 mM NaH2PO4, 450 mM NaCl, 1 mM

DTT, pH 8.0). Aliquots of the conjugate were shock frozen and stored at - 20 °C.

MALDI-TOF mass spectrometry

Mass spectra of proteins were recorded with an Autoflex speed MALDI-TOF mass

spectrometer (Bruker Daltonics). The matrix solution was created by dissolving 7.6 mg of 2’,5’-

dihydroxyacetophenone in 375 μl analytical ethanol and adding it to 125 μl of an 18 mg/ml

aqueous solution of ammonium hydrogen citrate. Proteins were desalted with the help of

Supel-Tips C18 (Sigma-Aldrich) and eluted in 2 µl of a 50:50-mixture (v/v) of acetonitrile and

0.1 % trifluoroacetic acid. 2 μl of 2 % TFA and 2 μl of matrix solution were added to the eluted

protein. 0.5 μl of this mixture was spotted on an MTP 384 ground steel target plate (Bruker

Daltonics) and allowed to dry. Samples were mounted onto a target frame and analyzed with

the standard methods LP_20-50kDa or RP_5 20kDa from the Bruker library.

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2. Electronic Supporting Figures

Fig. S1. Taspase1 is a type II asparaginase. a) Protein Substrate binding to a protease: Substrate

residues participating in binding to the protease (gray) are numbered P1-Pn (non-primed sites;

blue) N-terminal of the cleavage site and P1’-Pn’ (primed sites; red) C-terminal of the cleavage

site. The respective binding sites at the surface of the protease that accommodate the amino

acid side chains are numbered S1-Sn and S1’-Sn’, respectively. Cleavage occurs between the

P1 and P1’ position. b) Protein families: Human proteases can be classified according to their

active site into five classes that can be located either intra- (inner ring) or extracellular (outer

ring). Numbers inside the rings indicate the number of members of the color-coded classes.

Taspase1 is classified as an intracellular Threonine protease. Modified from Wuensch et al.,

2016.7 c) Taspase1 displays a conserved asparaginase fold. Overlay of the crystal structure of

Taspase1 (red; PDB 2a8j) with human asparaginase (4gdw), plant asparaginase (2gez), E. coli asparaginase (2zal) and F. meningosepticum glycosylasparaginase (1ayy) reveals a conserved

asparaginase fold. The structures were aligned using PyMOL. The close-up view of the active

site residues of Taspase1 (red) and its homologs (gray) shows a conserved side chain

orientation. Side chains are displayed as sticks and the catalytic threonine is highlighted in

yellow. d) Autocatalytic processing of Taspase1. The Taspase1 proenzyme comprises 420

amino acids and undergoes spontaneous autoproteolytic cleavage between Asp233 and

Thr234. This yields a 25 kDa a-subunit (amino acids 1-233; red) and a 20 kDa b-subunit (amino

acids 234-420; orange). In this process, Thr234 becomes the N-terminal amino acid of the b-

subunit and its free hydroxyl group renders Taspase1 proteolytically active. Two ab-

heterodimers assemble to a hetero-tetramer with abba-structure. N, amino-terminus; C,

carboxy-terminus.

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Fig. S2. Taspase1 protein purification. a) NiNTA affinity purification of wild-type Taspase1.

Chromatogram includes the gradient of elution buffer, and samples for SDS-PAGE were taken

from the supernatant (S) before NiNTA purification and from elution fractions indicated with

arrows. The corresponding gel reveals fractions containing full-length Taspase1 (fl), a-subunit

(a) and b-subunit (b). Fractions highlighted in blue were pure enough for gel filtration, while

the impure fractions highlighted in green were applied to another NiNTA affinity

chromatography. b) Preparative gel filtration of wild-type Taspase1.

The two peaks visible in the gel filtration chromatogram were analyzed by SDS-PAGE,

demonstrating that the second peak (highlighted in blue) contains pure full-length Taspase1,

a-subunit and b-subunit. c) Calibration plot for Superdex 200 10/300. Calibration was

performed with gel filtration buffer containing 450 mM NaCl. Ferritin (450 kDa), aldolase

(161 kDa), conalbumin (75 kDa), a-amylase (54 kDa) and ribonucleaseA (13.7 kDa) were used

as references. Elution volumes were plotted against the logarithmic molecular weight and

linear regression was performed. The dashed lines indicate the elution volume (14.5 ml) and

corresponding log(Mw) (2.01 = 102 kDa). d) NiNTA affinity purification of active Taspase1. The

His-tagged a-subunit and the untagged b-subunit were co-purified. Chromatogram includes

the elution gradient. The SDS gel separating proteins of the elution fractions shows that the

second peak (highlighted in blue) contains the a- and b-subunit with only few impurities

allowing subsequent gel filtration. e) Gel filtration of active Taspase1. The third peak in the

chromatogram (highlighted in blue) contains pure active Taspase1 as revealed by SDS-PAGE

of protein containing fractions. f) NiNTA affinity purification of inactive Taspase1. The fractions

highlighted in blue were used for gel filtration, while the fractions highlighted in green were

purified again by NiNTA affinity chromatography. g). Preparative gel filtration of inactive

Taspase1. The fraction highlighted in blue were concentrated, shock frozen and stored at -

20 °C. h) The SDS-PAGE gel shows inactive Taspase1 with minor impurities after NiNTA affinity

chromatography. The visible band near 45 kDa corresponds to the full-length enzyme.

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Fig. S3. Taspase1 secondary structure and stability. a) Protein Far-UV CD spectra of wild-type

Taspase1 (wt), active Taspase1 (with shortened loop) and inactive Taspase1 (D233A/T234A)

indicate a similar secondary structure composition of all three protein variants. The shape of

the spectrum, especially the minima at 222 nm and 208 nm hint at a helix-rich protein with

fractions of b-strand. b) Secondary structure deconvolution of the wt CD spectrum using the

CDSSTR algorithm confirm 55 % helix (blue), 23 % sheet (cyan), 11 % turn (light grey) and 10 %

random coil (dark grey) elements. Numbers indicate percent values. c) CD melting curves of

wild-type Taspase1 (wt Taspase1; black; Tm = 59 °C), active Taspase1 (blue; Tm = 60 °C) and

inactive Taspase1 (red; Tm = 63 °C) in 50 mM phosphate buffer. d) Tryptophan fluorescence

melting curves show that wild-type Taspase1 in 50 mM phosphate buffer (black; Tm = 56 °C)

can be stabilized by addition of 10 % sucrose (blue; Tm = 73 °C) or 450 mM NaCl (red;

Tm = 77 °C). e) Protein Far-UV CD spectra in the presence of nanoparticles with a diameter of

20 nm (Amsil20) show no change in secondary structure content of Taspase1 at nanoparticle

concentrations between 0 and 150 µg/ml (cyan).

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Fig. S4. Taspase1 molecular weight, multimerization and autocatalytic processing. a) MALDI-

TOF mass spectra of wild-type Taspase1 (top panel), active Taspase1 (middle panel) and 15N-

labeled inactive Taspase1 (lower panel) were recorded in the range of 15 to 50 kDa. The

arbitrary intensity units were rescaled. Arrows indicate masses of the respective full-length

protein (fl), a-subunit (a), b-subunit (b), and shortened a-subunits (amino acids 1-206 or 1-

195). For improved clarity, double and triple charged masses are not labeled. b/c) Analytical

gel filtration chromatograms of a calibrated Superdex 200 column reveal that wild-type

Taspase1 (b) and inactive Taspase1 (c) elute in the range of 90-110 kDa (labeled T for tetramer),

corresponding to a hetero-tetramer. At the expected dimer size of 45-55 kDa (labeled D for

dimer), no peak is visible. d) Autocatalytic processing of Taspase1. 10 µM wild-type Taspase1

were incubated in gel filtration buffer at 37 °C, and samples were taken for SDS-PAGE (15 %)

at indicated time points. The expected sizes of full-length Taspase1, the a- and b-subunit are

marked with arrows. No autocatalytic processing was observed for the inactive Taspase1

mutant even after 7 days. Lines indicated lanes from different gel runs.

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Fig. S5. Taspase1 proteolytic activity. a) Principle of the fluorogenic Taspase1 activity assay.

The model substrate contains the cleavage site for Taspase1, an N-terminal anthranilic acid

(Abz) and a C-terminal dinitrophenol moiety (DNP). After excitation of the Abz dye, the energy

is transferred to the DNP and the fluorescence is quenched. Upon cleavage by Taspase1, dye

and quencher are separated. Hence, Abz is no longer quenched and can emit photons. b) In

vitro Taspase1 activity assay. Emission of the fluorogenic substrate increases over time in

presence of Taspase1. The initial rate and plateau at substrate depletion increase with

substrate concentration. Substrate with mutated cleavage site (red) shows no increase in

fluorescence intensity. c) Loss of Taspase1 activity over time. Incubation of wild-type Taspase1

at 37 °C in measurement buffer containing 10 % sucrose shows a loss of activity over time.

Nonlinear fit yields a half-life around 2.5 h. d) Activity of the Taspase1 variants. Specific activity

of wild-type Taspase1 (wt, black, 0.086 ± 0.0009 μmol * min-1 * mg-1), active Taspase1

(shortened loop, blue, 0.063 ± 0.002 μmol * min-1 * mg-1), inactive Taspase1 (D233A/T234A

mutant, 0.00003 μmol * min-1 * mg-1) and buffer control (no Taspase1, 0.00006 μmol * min-

1 * mg-1) in the presence of 8 μM substrate. For the inactive mutant and the buffer control no

activity was observed. Error bars indicate standard deviations. e) Substrate specificity of

Taspase1. Michaelis-Menten plots at 37 °C for the two Taspase1 peptides with the cleavage

sites CS1 (blue squares) and CS2 (black circles) of the MLL protein. Catalytic parameters

obtained by nonlinear fitting can be found in Table S6 (Supporting Information). f) Specific

activity of eukaryotic Taspase1 in human HeLa cell lysates. Assay was performed at 30 °C in

the presence of 8 μM substrate in the form of either Hela lysate with overexpressed Taspase1-

GFP fusion protein (transfected) and purified Taspase1 after heterologous bacterial

expression (bacterial). Both Taspase1 species show a similar specific activity.

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Fig. S6. Structural models of Taspase1. a) Structure of the dimer of Taspase1 showing the two

monomeric molecules (dark and light grey, respectively) as a ribbon representation projected

onto the ghost surface (white). The C-termini of Taspase1 are located 60 Å apart at the

opposite ends of the dimer (indicated by the arrows). b) Visualization of surface patches on

the dimer surface as presented in (a). The front view was additionally rotated as indicated to

allow views on the bottom and back site of the molecule as well, including the positively

charged active site. Color code: from negative charge (red) to positive charge (blue) as

visualized by the open-source web browser application NGL viewer

(http://nglviewer.org/ngl/). Charges were mapped on a model based on PDB 2a8j. c) Interface

of the two Taspase1 subunits. The a-subunit possesses a positively charged patch (blue) at

the interface to the b-subunit (yellow). Conversely, the b-subunit shows a negatively charged

patch (red), which allows interaction with the a-subunit (yellow). Color code: from negative

charge (red) to positive charge (blue) as visualized by Swiss-Model

(https://swissmodel.expasy.org). Charges were mapped on a model based on PDB 2a8j.

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3. Electronic Supporting Tables

Table S1. Nanoparticle characterization.

Nanoparticle TEM Radius ±SD [nm]

DLS Hydrodynamic radius [nm]

Zeta potential ζ ± SD 8

AmSil8 9.6 ± 2.2 12.5 ± 0.19 -11 ± 5

AmSil20 15.7 ± 1.9 17.6 ± 0.1 -25 ± 6

Amsil125 54.9 ± 17.2 71.3 ± 0.05 -32 ± 4

Table S2. Parameters used for anisotropy measurements.

Parameter Atto488 Anthranilic acid

Excitation wavelength 501 nm 320 nm

Emission wavelengths 523 nm 420 nm

Excitation slit width 10 nm 20 nm

Emission slit width 20 nm 20 nm

PMT voltage 400 V 600 V

Average time 5 s 5 s

Temperature 20 °C 20 °C

G factor 1.6724 1.1373

Concentration 1 µM 1 µM

Table S3. Parameters used for kinetic measurements with the fluorogenic assay.

Parameter Value

Excitation wavelength 320 nm

Emission wavelengths 420 nm

Excitation slit width 20 nm

Emission slit width 20 nm

PMT voltage 480 V

Average time 5 s

Temperature 37 °C

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Table S4. Parameters used for recording of far-UV CD spectra.

Far-UV spectra Melting curves

Parameter Value Parameter Value

Start 260 Wavelength 225 nm

End 200 Temperature slope 1 °C/min

Scanning mode Continuous Delay time 180 s

Response time 0.5 sec Response time 8 s

Bandwidth 2 nm Bandwidth 1 nm

Data pitch 0.2 nm Data pitch 0.1 °C

Temperature 21 °C Temperature 20-85 °C

Accumulations 20

Scanning speed 100 nm/min

Table S5. Parameters used for recording of fluorescence melting curves.

Parameter Value

Excitation wavelength 280 nm

Emission wavelengths 334 nm and 376 nm

Excitation slit width 20 nm

Emission slit width 10 nm

PMT voltage 480 V

Average time 5 s

Hold time 180 s

Temperature slope 0.5 °C/min

Data interval 0.1 °C

Temperature range 20-95 °C

Table S6. Catalytic parameters of Taspase1 target sequences at 37 °C.

MLL cleavage site CS1 [Mca]-GKGQVDGADDK-[DNP]a)

CS2 [Abz]-KISQLDGVDDK-[DNP]a)

Km (μM) 18.4 ± 2.8 2.7 ± 0.1

vmax (µmol min-1 mg-1) 0.03 ± 0.003 0.11 ± 0.002

kcat (s-1) 0.0274 0.083

kcat/Km (l mol-1 s-1) 1487 31190

a)[Mca]: 7-amino-4-methylcoumarin; [DNP]: dinitrophenol; [Abz]: anthranilic acid;

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Table S7. Channel settings used for fluorescence microscopy.

Channel Excitation wavelength

Emission wavelength

Exposure time

Hoechst 345 nm 455 nm 25 ms

GFP 480 nm 510 nm 500 ms

mCherry 545 nm 610 nm 500 ms

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