electronic supporting information(biotasp, figure 4b), serving as fluorescent reporter substrate...
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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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