Chemical zymogens for the protein cysteinome | Panda Anku

All chemicals and proteins, unless stated otherwise, were purchased from Sigma Aldrich and used without purification. Deuterated solvents were supplied from Euriso-Top. Ultrapure water was dispensed from MilliQ Direct 8 (Millipore) [18.2 MΩ •cm].

Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury 400 MHz spectrometer, running at 400 MHz. Chemical shifts (δ) are reported in ppm relative to the residual solvent.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were recorded using a Bruker Autoflex II MS with nitrogen laser (337 nm) in linear positive 20–200 kDa mode. At least 100 laser shots covering the complete spot were accumulated for each spectrum. For molecular weight determination, sinapinic acid (20 g/L) in 50 % acetonitrile with 0.1% trifluoroacetic acid was used as matrix. Sample solution (0.1 to 1.0 g/L) was mixed with an equal volume of matrix and 4 μL of the resulting mixture was loaded onto a ground steel target plate and allowed to dry for cocrystallization.

Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) was performed on a system containing an Agilent 1260 Infinity II Isocratic HPLC Pump, a Wyatt miniDAWN 3-angle static light scattering detector, an Agilent 1260 Infinity II VWD UV–Vis detector, and a Shodex RI 501 refractive index detector. The system was equipped with a Superdex 200 Increase 10/300 GL column from GE Healthcare with a length of 300 mm, an internal diameter of 10 mm, and 8.6 µm particle size. This provided an effective molecular weight range of 10,000–600,000. The eluent was 0.01 M PBS with 10% methanol and it ran at a temperature of 30 °C and a flow rate of 0.75 mL/min. Molar mass analyses were conducted using ASTRA® Software Basic.

Reverse-phase HPLC analyses were performed on an Agilent apparatus equipped with a C18 column with 2.7 μm particles, a length of 150 mm and an internal diameter of 3.0 mm from Supelco Analytical. HPLC mobile phase A was ultrapure H2O supplemented with 0.1% TFA (v/v) and mobile phase B acetonitrile supplemented with 0.1% TFA (v/v). Elution was performed starting with solvent B 5% to B 100% over 15 min, hold B 100% for 4 min at T = 40 °C and a flow rate of 0.4 mL/min. Detection was performed by UV detector (220 nm and 280 nm).

Fluorescence and luminescence measurements were recorded on an Enspire 2300 Multilabel Reader (Perkin Elmer®) or a SynergyH1 microplate reader (BioTek®)

Preparation of compounds

Solution-based ring-opening polymerization (ROP) of lipoic acid

In a typical procedure, α-lipoic acid (300 mg, 1.5 mmol, 1.0 equiv.) and 2-iodoacetamide (5.4 mg, 29.1 μmol, 0.02 equiv.) was added to a 5 mL pre-dried pear-shaped flask and dissolved in 100 μL dry DMF, heated to 70 °C and stirred magnetically under and argon atmosphere for 2 h to yield a dark-brown solution. Hereafter, NaHCO3 (pH 8.5, 0.4 M) was added to the reaction mixture to deprotonate the carboxylic acid thereby solubilizing the polymer. A spatula was used to release the stirring bar from the glass flask and the reaction mixture was heated to 40 °C to speed the process. The resulting solution was transferred to a dialysis membrane (MWCO 3.5 kDa) and dialyzed against water. The polymer was recovered via freeze-drying as a white fluffy solid (166 mg, 55%). 1H-NMR (400 MHz, D2O), δ, ppm: 3.29 (s, 4H), 2.98–2.85 (m, 59H), 2.36–1.98 (m, 84H), 1.77–1.36 (m, 122H). Mn (NMR) = 4.2 kDa, Mn (SEC-MALS) = 9.9 kDa, Đ = 1.3

Reduction of papain and bromelain

Papain and bromelain, as cysteine proteases, are commercially provided as partially inactivated enzymes. Activation is accomplished by incubation with a thiol-containing compound or a reducing agent. In a typical experiment, to a solution of papain (5 g/L, 0.17 mM, 0.5 mL) in 50 mM phosphate buffer pH 6.8 with 10 mM EDTA was added tris (2-carboxyethyl) phosphine hydrochloride (TCEP, 1.1 mM, 5 equiv). The reaction was incubated at 37 °C for 1 h. After this time, TCEP was removed by gel filtration (NAP-5, GE Healthcare) in the same buffer and concentrated using spin filtration (MWCO 3 kDa).

Preparation of PEG-S-S-TP

A solution was prepared of 2,2’-dithiodipyridine (45.8 mg, 4.0 equiv.) and acetic acid (1.5 μL, 0.5 equiv.) in methanol (7.5 mL) under an atmosphere of argon. MeO-PEG-SH-6000 (312.0 mg, 1.0 equiv.) was dissolved in a mixture of methanol:acetic buffer 50 mM (pH = 4.5) 1:1 v:v (2 mL), purged with argon and added dropwise to the reaction mixture. The reaction was left stirring at room temperature for 21 h. The product was precipitated twice into cold diethyl ether followed by centrifugation. The precipitated product was obtained by filtration followed by solvent removal in vacuo to yield PEG-S-S-TP (176.0 mg, 53%) as a white solid. 1H-NMR (400 MHz, Methylene Chloride-d2) δ 8.45–8.41 (m, 1H), 7.83–7.78 (m, 1H), 7.71–7.65 (m, 1H), 7.13–7.07 (m, 1H), 3.77 (t, J = 5.2 Hz, 2H), 3.71–3.41 (m, 144H), 3.34 (s, 3H), 3.00 (t, J = 6.0 Hz, 2H).

General protocol for zero-length zymogens

Reduced papain, bromelain (2 g/L) or creatine kinase (1 g/L) were reacted with S-methyl methanethiosulfonate (MMTS, 500 equiv.) in sodium phosphate buffer (50 mM, 10 mM EDTA, pH 6.8). The solution was stirred at 4 oC overnight and the proteins were purified/buffer exchanged to sodium acetate buffer (2.5 mM, pH 4.5, 50 mM NaCl) using Amicon centrifugal filters (MWCO 3 kDa).

General protocol for PEG-based zymogens

Reduced papain and bromelain (100 μM) or creatine kinase (25 μM) were combined with 10 eq. of PEG-TP (synthetized as described above or commercially available MeO-PEG-OPSS 5 kDa from Iris Biotech) in acetate buffer (2.5 mM, pH 4.5, 50 mM NaCl) and incubated at 37 oC for 1 h. After this time, the crude sample was purified by gel filtration (NAP-5) in the same buffer.

General “chain transfer” protocol for preparation of LA PDS zymogens

Reduced papain or bromelain (2 mg/mL) and excess LA PDS (10×, by weight) were dissolved in sodium phosphate buffer (50 mM, 10 mM EDTA, pH 6.8). The solution was stirred at 37 °C for 2 h. After this time, the crude reactions were purified by Amicon centrifugal filtration in the same buffer.

General “grafting from” polymerization protocol for preparation of LA PDS zymogens

Reduced papain, bromelain (0.5 g/L) or creatine kinase (1 g/L) were dissolved in solutions containing 100 or 25 mM of LA in 0.2 M NaHCO3, respectively, and 1 mL of these reaction solutions was kept in the freezer at −20 oC for 2 h. After this time, 200 μL of a 0.1 M solution of iodoacetamide in Milli Q water was added to the frozen reactions and left stirring at room temperature for 30 min. The zymogens were purified by gel filtration (CentriPure P10) in milli Q water.

Preparation of fluorescently labeled, self-quenched albumin

BSA and potassium carbonate were dissolved in milli Q water to 10 g/L each. Then fluorescein isothiocyanate (FITC) was added to the solution from a concentrated DMSO stock to a final concentration of 2 g/L (final DMSO 2% v/v). The reaction was left at 37 oC under stirring for 21 h and purified extensively by Amicon centrifugal filtration (MWCO 3 kDa) in Milli Q water until no absorbance from fluorescein could be observed in the wash waters.

Preparation of Cys-blocked BSA

BSA was dissolved in borate buffer (25 mM, pH 8.0) to a final concentration of 2 g/L. To that solution 350 equivalents of iodoactamide were added and the mixture was incubated at 37 oC for 30 min. After that time, the sample was purified by gel filtration (CentriPure P10) in Milli Q water.

Experimental protocols

RP-HPLC analysis of poly (lipoic acid) degradation using dithiothreitol (DTT)

Poly (lipoic acid) was incubated at room temperature in the presence and absence of dithiothreitol in 50 mM phosphate buffer, pH 8.0. At specific time points (t = 5 min and 24 h), reactions were analyzed by RP-HPLC. Lipoic acid (0.10 g/L, 0.5 μmol) incubated under the same conditions served as control.

Reactivation of papain LA PDS zymogens

Solutions of 1 μM of papain and papain LA PDS zymogens prepared by either chain transfer or polymerization methods were incubated with 10 μM Nα-benzoyl-L-arginine-7-amido-4-methylcoumarin (AMC) in phosphate buffer (50 mM, 10 mM EDTA, pH 6.8). To each sample was added DTT to 10 mM or just buffer as a control with final reaction volumes of 100 μL. After 60 min at 37 oC substrate hydrolysis was measured by recording fluorescence at λex/λem 370/460 nm.

Effect of different polymerization quenchers on LA PDS zymogen reactivation

LA PDS zymogens were prepared by polymerization as described above, but changing the quenching reagent used after polymerization. The quenchers used were: iodoacetamide, 2,2’dithiodipyridine, 4-maleimidobutyric acid and phenyl vinyl sulfone in concentrations of 0.1 M. A reaction in the absence of quencher was also prepared. After purification, equal volumes of each reaction were diluted in borate buffer (25 mM, pH 8.0) to a final protein concentration of 2 μM, combined with 5 μM of AMC. Catalytic activity in the presence or absence of DTT (2 mM) was analyzed by substrate hydrolysis in 100 μL reaction volumes as detected by fluorescence increase at λex/λem 370/460 nm at 1 min intervals for 1 h at 37 oC in a plate reader.

Effect of LA concentration during polymerization

LA PDS zymogens of papain were prepared using the “grafting from” polymerization protocol described above with concentrations of LA during the reaction set to 100 or 25 mM and also a control without LA. After quenching of the reactions with iodoacetamide and purification, equal volumes of each reaction were diluted in borate buffer (25 mM, pH 8.0) to 0.3 μM protein (as determined by UV absorbance at 280 nm), combined with 10 μM of AMC. Catalytic activity in the presence and absence of DTT (2 mM) was analyzed by substrate hydrolysis in 100 μL reaction volumes as detected by fluorescence increase at λex/λem 370/460 nm at 1 min intervals for 2 h at 37 oC in a plate reader. These experiments were performed in three independent replicates (independent zymogen syntheses).

Effect of pH on the reactivation of papain LA PDS zymogens

Papain LA PDS zymogen prepared by polymerization method as in section 2.7 was dissolved at 5 μM and combined with 50 μM AMC in different buffers with the addition of 10 mM DTT. Samples wihout DTT were prepared as controls. The different buffers were: formic acid (20 mM, pH 4.0), 2-(N-morpholino)ethanesulfonic acid (MOPS, 20 mM, pH 6.0), phosphate buffer (20 mM, pH 7.0) and borate buffer (25 mM, pH 8.0). Substrate hydrolysis was measured by recording fluorescence at λex/λem 370/460 nm at 1 min intervals for 2 h at 37 oC in a plate reader. These experiments were performed in 3 independent replicates (independent zymogen syntheses).

Reactivation studies of papain and bromelain zymogens

Reactivation of different zymogens (Z0, ZPEG, and ZLA) of papain and bromelain was studied by monitoring fluorescence increase upon hydrolysis of AMC. Briefly, solutions containing papain zymogens (Z0, ZPEG = 1 μM, ZLA = 0.3 μM as determined by UV absorbance at 280 nm) or bromelain zymogens (Z0, ZPEG, ZLA = 5 μM, as determined by UV absorbance at 280 nm) were combined with 10 μM of substrate and DTT (2 mM) in borate buffer (25 mM, pH 8.0) in a volume of 100 μL. Samples without DTT were used as controls. Fluorescence (λex/λem 370/460 nm) was monitored at 1 min intervals at 37 oC in a plate reader. These experiments were performed in three independent replicates (independent zymogen syntheses).

Reactivation studies of creatine kinase zymogens

Reactivation of different zymogens (Z0, ZPEG, and ZLA) of creatine kinase (CK) was quantified via a coupled bi-enzymatic assay. Briefly, unmodified CK or zymogens were diluted to 100 nM (as estimated by a protein assay with fluorescamine using CK as reference) in Tris-HCl buffer (20 mM, pH 8.0) in a white microplate. Then, DTT was added, and quickly after, a solution containing ADP, creatine phosphate (CP), luciferin and Cell-Titer Glo® 2.0 Cell Viability Assay luciferase (Promega) was added to the wells. Luminescence was monitored at 37 oC in regular time intervals over 1 h (RLU integrated every 2 s) in a plate reader. Final volume in every well was 100 μL and concentration of reagents was as follows: DTT 20 μM, ADP, CP and luciferin 1 μM, Cell-Titer Glo® 2.0 1:50 dilution (v/v). Controls included samples of each zymogen/protein without DTT, CK without CP and samples without CK or zymogen. At the end-point, luminescence was also imaged inside an ImageQuantTM LAS 4000 camera system (GE Healthcare) with 3 s exposure. These experiments were performed in three independent replicates (independent zymogen syntheses).

Gel electrophoresis of creatine kinase zymogens

Samples of 2 μg of creatine kinase and creatine kinase zymogens were prepared in Milli Q water supplemented with LDS buffer (Pierce, 1:4 final dilution) and NuPAGETM sample reducing agent (in a final dilution of 1:8). For each protein or zymogen, a sample without reducing agent was also prepared. Samples were incubated 20 min at 37 oC and subsequently 10 μL were loaded into a NuPAGETM gel (4 to 12% bis-tris, 1.0 mm) and run for 50 min in MOPS buffer at 150 V and later stained with coomassie blue.

Reactivation of papain zymogens with different protein activators

Reactivation of different papain zymogens (Z0, ZPEG, and ZLA) was studied by monitoring fluorescence increase upon hydrolysis of AMC. Briefly, solutions containing papain zymogens (Z0, ZPEG = 1 μM, ZLA = 0.3 μM as determined by UV absorbance at 280 nm) were combined with 10 μM of substrate and creatine kinase (CK) or pyruvate kinase VII at 1 μM in borate buffer (25 mM, pH 8.0). A negative control with 1 μM lysozyme (non-thiol-containing protein) was prepared in the same conditions as well. Reactivation was also performed with transglutaminase and pyruvate kinase II at 1 μM, but with a 10-fold increase in zymogen content (Z0, ZPEG = 10 μM, ZLA = 3 μM). Fluorescence (λex/λem 370/460 nm) was monitored at 1 min intervals at 37 oC in a plate reader. All well volumes were set to 100 μL and samples with DTT (2 mM) or without any activator were prepared as positive and negative controls, respectively. These experiments were performed in three independent replicates (independent zymogen syntheses).

Reactivation of papain zymogens by protein activator with degradation of a protein substrate

In a 96-well plate, solution of self-quenched BSA-FITC in borate buffer (25 mM, pH 8.0) was combined with different papain zymogens (Z0, ZPEG = 1 μM, ZLA = 0.3 μM as determined by UV absorbance at 280 nm) and 1 μM of creatine kinase in the same buffer. Reaction volume was 100 μL; samples with DTT (2 mM) and no activator were used as positive and negative control, respectively. Progress of the reaction was observed by the increase in fluorescence λex/λem 490/520 at 37 oC in a plate reader for 2 h in intervals of 1 min. After 2 h the plate was imaged inside an ImageQuantTM LAS 4000 camera system (GE Healthcare) using the GFP filter in fluorescence mode. These experiments were performed in three independent replicates (independent zymogen syntheses).

Reactivation of papain zymogens via protein–protein interaction imaged using SDS PAGE

Samples with 4 μM of Cys-blocked BSA and 1 equivalent of different papain zymogens (Z0, ZLA) or pristine papain were prepared in Milli Q water and supplemented with SDS buffer (Pierce, 1:4 final dilution). For each protein or zymogen, a sample with 3.3 μM PKVII was also prepared. As positive controls, one sample containing only 4 μM of quenched BSA, and another with 3.3 μM PKVII were prepared. Samples were incubated 10 min at room temperature and subsequently 10 μL were loaded into a NuPAGETM gel (4 to 12% bis-tris, 1.0 mm) and run for 55 min in MOPS buffer at 150 V and later stained with coomassie blue.

Rate of chain transfer reaction

Rate of chain transfer reaction was studied by monitoring release of the polymer end-cap, in this case thiopyridine, upon triggered depolymerization by papain. In a 96-well plate, a solution of 1.2 g/L LA PDS end-capped with thiopyridine was combined with 6 μM papain in borate buffer (25 mM, pH 8.0). Absorbance at 343 nm was monitored at 10 s intervals at 37 oC in a plate reader, and later translated to thiopyiridine concentration by using a calibration curve. All well volumes were set to 100 μL and samples with DTT (5 mM) or with the polymer alone were prepared as positive and negative controls, respectively. These experiments were performed in three independent replicates.

Activity read-out for zymogen exchange experiment with CK

Samples containing 10 μM (determined by UV absorbance at 280 nm) of papain zymogen (ZLA), 27 μM papain inhibitor (E-64, Sigma), and 4 μM creatine kinase were prepared. First, papain zymogen was incubated with the inhibitor at room temperature for 30 min, and then CK was added and the mixture was incubated for 1 h more at 37 oC. After this time, samples were dissolved in Tris-HCl buffer (20 mM, pH 8.0) in a white 96-well plate to a concentration of 0.1 μM of CK. Samples with 0.1 μM CK and 0.3 μΜ papain zymogen were prepared as positive and negative controls, respectively. Immediately after, CK substrates ADP, CP and luciferin at a concentration of 1 μM and Cell-Titer Glo® reagent in a 1:10 dilution were added (final volume in well 100 μL) and luminescence was monitored at 37 oC in regular time intervals 1 h (RLU integrated every 2 s) in a plate reader. These experiments were performed in three independent replicates (independent zymogen syntheses).

Zymogen exchange experiment with different proteins

Samples with papain-LA PDS zymogen at a concentration of 8.3 μM (determined by UV absorbance at 280 nm) were incubated with 2 equivalents of papain inhibitor (E-64, Sigma) at room temperature for 30 min. Then, 3.5 μM of different protein activators (creatine kinase, transglutaminase and pyruvate kinase VII) were added and incubated for 1 h more at 37 oC. After this time samples were analyzed in MALDI-TOF-MS, together with the corresponding pristine proteins diluted in Milli Q (papain, creatine kinase, transglutaminase and pyruvate kinase VII) as controls.

Trans-PEGylation from papain Z
PEG to creatine kinase

Sample containing 72 μM of papain ZPEG (determined by UV absorbance at 280 nm) was incubated with 2 equivalents of papain inhibitor (E-64, Sigma) at room temperature for 30 min. Then, 5 μM of creatine kinase was added and incubated for 1 h more at 37 oC. Controls with pristine papain or creatine kinase diluted in Milli Q were prepared as well. All samples were analyzed in MALDI-TOF-MS.

Zymogen exchange experiment with two protein acceptors

Samples with papain-LA PDS zymogen at a concentration of 8.3 μM (determined by UV absorbance at 280 nm) were incubated with 2 equivalents of papain inhibitor (E-64, Sigma) at room temperature for 30 min. Then, 3.5 μM of creatine kinase or pyruvate kinase VII were added and incubated overnight. The next day, 1 μM of pyruvate kinase or creatine kinase, respectively, were added. Samples without the second protein addition and controls with pristine creatine kinase and pyruvate kinase diluted in Milli Q were prepared as well. Al samples were analyzed in MALDI-TOF-MS.

Imaging the two-step zymogen exchange reactions

Samples with papain-LA PDS zymogen at a concentration of 15 μM (determined by UV absorbance at 280 nm) were incubated with 3.5 equivalents of papain inhibitor (E-64, Sigma) at room temperature for 30 min. Then, 15 μM of creatine kinase were added and incubated for 1 h at 37 oC. After that time, the reaction was quenched with 20 mM iodoacetamide for 30’ and then purified by gel filtration (CentriPure P10) in borate buffer (25 mM, pH 8.0). This sample was then transferred to a 96-well plate (per triplicate), with and without the addition of 5 μM of pyruvate kinase and incubated for 1 h more (samples with the second protein addition were first mixed with 3.5 equivalents of papain inhibitor E-64). Controls with pristine creatine kinase, papain-LA PDS zymogen and 5 μM pyruvate kinase diluted in borate buffer were prepared as well. Finally, CK substrates ADP, CP and luciferin at a concentration of 1 μM and Cell-Titer Glo® reagent in a 1:10 dilution were added and, after 40 min reaction, luminescence was imaged inside an ImageQuantTM LAS 4000 camera system (GE Healthcare) with 8 s exposure.

Analyses

Statistical analysis

Where reported, statistical significance was evaluated with a two-way ANOVA with the Sidak’s multiple comparisons test performed in the software Graphpad Prism®.

Cysteine surface accessibility analysis

The tertiary structures of glutathione reductase, pyruvate kinase, transglutaminase, protein kinase A and polynucleotide kinase were downloaded from the Protein Data Bank (PDB codes: 2HQM, 3N25, 1KV3, 4WB5, 1AQF, and 1LTQ, respectively). Molecular graphics and solvent accessible surface areas (Å2) calculations of cysteine residues were performed with UCSF Chimera (Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311). Accessible surface area values were determined with a probe radius of 1.4 Å, equivalent to the radius of a water molecule. As a reference for cysteine accessibility, calculated values were compared to reported accessible surface area of a cysteine residue in a folded protein (5 Å2)46. In this analysis, all cysteine residues with calculated values above 5 Å2 were considered accessible.

Prediction of cysteine pKa and estimation of partial atomic charges in lipoic acid

The pKa of cysteinyl thiol groups in protein activators was predicted using PROPKA. The PROPKA method consists of an empirical approach to determining pKa values by calculating the effect of the protein environment on an amino acid side chain47,48. For the thiol group in cysteine residues, the starting pKa in the calculations is 9.0. Poisson-Boltzmann electrostatic surface map for a lipoic acid trimer was generated with Maestro (Release 2022-1: Maestro v.13.1.137, Schrödinger, LLC, New York, NY, 2021). The trimer of lipoic acid provided a closer representation to poly (lipoic acid) without extensive molecular dynamic simulations. Partial atomic charges for the thiols in lipoic acid were determined using Discovery Studio (BIOVIA, Dassault Systèmes, Discovery Studio Visualizer, v21.1.0.20298, San Diego: Dassault Systèmes, 2020) for comparison to cysteinyl thiol pKa values.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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