We designed four ArgRS mutants including D118A, D317A, Y313A, R324A based on ArgRS structure. The mutation was introduced into pET22b/ArgRS using the site-directed mutagenesis and purified these proteins with the same method as described previously. The formation constant and thermodynamic parameters for the inclusion of Arginine in ArgRS were measured by the titration calorimetry method by using an ITC MicroCal 200 (GE Life Sciences). All solutions were prepared in a 200 mmol/L Tris-HCl pH 7.5, 300 mmol/L sodium chloride. A solution (0.25 mmol/L) of ArgRS was placed in the sample cell, and a 2.5 mmol/L solution of Arginine was added in a series of 20 injections, the heat evolved was recorded at 25°C. The heat of injecting the Arginine into a neat buffer solution is nearly zero. The data were analyzed and the binding isotherm was fitted to a single-site model in the ORIGIN 7.0 software (GE Life Sciences).
Origin 7
Manufactured by GE Healthcare
Sourced in United States
Origin 7.0 is a laboratory equipment product from GE Healthcare. It is designed for core analytical functions. The product specifications and technical details are not available for an unbiased and factual description at this time.
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Lab products found in correlation
1 mm quartz cuvette, by Hellma (2 mentions)
Spectra analysis v 2, by Jasco (2 mentions)
Nanodrop instrument, by Thermo Fisher Scientific (2 mentions)
J 1500 spectropolarimeter, by Jasco (2 mentions)
Itc200, by GE Healthcare (2 mentions)
Itc200 instrument, by GE Healthcare (1 mentions)
Pnp glcnac, by Merck Group (1 mentions)
Vp itc calorimeter, by GE Healthcare (1 mentions)
Chitobiose, by Merck Group (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Microcal peaq itc, by Malvern Panalytical (1 mentions)
Itc200 calorimeter, by GE Healthcare (1 mentions)
Auto itc200 microcalorimeter, by GE Healthcare (1 mentions)
25 protocols using origin 7
1
Arginine Binding Kinetics in ArgRS
2
ITC Study of MG and Mb Variants
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Isothermal titration calorimetry (ITC) study of MG and F43H/H64A Mb was performed using Microcal VP-ITC (GE life sciences) at 25 °C. Both F43H/H64A Mb (40 μM in 100 mM KPi buffer, pH 6.0) and MG (0.5 mM in the same buffer) were degassed in the ThermoVal instrument (Microcal). For the titration experiment, 1.42 mL of F43H/H64A Mb solution was placed in the reaction cell, and the solution of MG was injected over 20 s with a total of 25 injections (2 μL for the first injection and 10 μL for the subsequent injections), with an interval of 150 s between each injection. The reaction cell was continuously stirred at a speed of 437 rpm, and the thermal change was recorded at 25 °C. The combined isotherm was fitted to a single point model in Origin 7.0 software (GE life sciences).
3
Circular Dichroism Spectroscopy of Proteins
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The experiments were performed as described in [68 (link)]. CD spectra were recorded using a J‐1500 spectropolarimeter (Jasco, Easton, MD, USA) equipped with a Peltier element for temperature control and a six‐position cuvette holder. Proteins were dialyzed in 5 mM MOPS pH:7.5; 5 mM NaCl, for 15 h, at 4 °C; 3× changes; constant stirring. Aggregated material was removed by centrifugation (20,000 g; 15 min; 4 °C) before protein concentration was determined on a Nanodrop instrument (280 nm; 2000 series; Thermo). The molecular extinction coefficient and molecular weight for A280 analysis was determined using the Expasy server (http://web.expasy.org/protparam/ ). Wavelength scan measurements (190–260 nm) were performed with 15 μM protein, at 20 °C; in 5 mM MOPS pH 7.5; 5 mM NaCl, using 1 mm quartz cuvettes (Hellma, Müllheim, Germany); data pitch: 0.5 nm; bandwidth: 1 nm; scanning speed: 50 nm·min−1; DIT: 0.5 s; accumulation: 3. Variable temperature measurements (15–90 °C) were performed with 15 μM protein; in 5 mM MOPS pH 7.5; 5 mM NaCl, using 1 mm quartz cuvettes (Hellma); interval 0.5 °C; gradient 1 °C· min−1; DIT: 0.5 s; bandwidth: 1 nm. Data were analyzed using the spectra analysis v.2 software (Jasco); Tmapp were derived by acquiring the first derivatives of the melting curves, using the calculus function of the Origin 7 software (GE Healthcare).
4
Calorimetric analysis of P-cadherin-antibody
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Interactions between antibodies and human P-cadherin were examined by means of ITC using an iTC200 instrument (GE healthcare). The sample cell of the calorimeter was filled with antibodies at a concentration range of 15–30 μM. TSP7 was titrated with cadherins at concentrations ranging from 150 to 300 μM. Measurements were performed at 15 °C. Data were analyzed with ORIGIN7 software (GE healthcare) using a one-site binding model.
5
Thermodynamic Analysis of PirA-PirB Interaction
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After the N-terminal tagged His10-PirAvp and the C-terminal tagged His6-PirBvp were both dialyzed against the same reaction buffer (30 mM Tris, pH8.0, 100 mM NaCl, 5% glycerol, 1mM DTT), they were respectively loaded into the sample cell and syringe of an iTC200 (GE Healthcare) instrument. For titration, 2 μL of 1100 μM PirBvp was injected into the cell containing 110 μM PirAvp every 150 s for a total of 19 injections at 25 °C. All data except the first injection point were included to calculate the thermodynamic parameters. The enthalpy (ΔH), binding entropy (ΔS), equilibrium constant (1/Kd), and stoichiometric ratio (N) were obtained by using the computer software ORIGIN 7 (GE Healthcare, Chicago, IL, USA, 2002). Gibbs free energy (ΔG) was calculated using the equation: ΔG =ΔH − TΔS.
6
ITC Analysis of HU-DMP19 Binding
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ITC200 (GE Healthcare) was used to measure the binding affinity of HU and DMP19. Before the titration, both HU and DMP19 were dialyzed against an ITC buffer of 20 mM Tris pH 7.4 and 50 mM NaCl. After the buffer change, the C-terminal His6-tagged Neisseria HU dimer (300 μM) and DMP19 monomer (30 μM) were loaded into the syringe and sample cell, respectively. Two μl of the HU was injected every 3 min until the DMP19 was saturated (a total of 20 injections) at 25°C. Except for the first injection point, data from the entire titration curve were used to calculate the thermodynamic parameters. The program ORIGIN 7 (GE Healthcare) was used to calculate enthalpy (ΔH), binding entropy (ΔS), the equilibrium constant (1/Kd) and the stoichiometric ratio (N). Gibbs free energy (ΔG) was calculated using the equation: ΔG = ΔH − TΔS.
7
Isothermal Titration Calorimetry of Protein-Compound Interactions
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ITC experiments were performed using a MicroCal AutoiTC200 (GE Healthcare). In a typical run, 25 automated injections of 1.65 μl with 170-s breaks between injections were made at 25°C with 600 rpm stirring speed on low feedback mode. The protein concentrations in the sample cell were varied between 25 and 100 μM, while the compound concentration in the syringe was varied from 300 to 800 μM. The buffer for both protein and compound solutions was the same as in the final step of the purification. The compounds were dissolved in DMSO, and then diluted with buffer. DMSO content in the final compound solutions did not exceed 0.5% (v/v). Data integration, fitting, and evaluation were performed using the software Origin 7 with the ITC200 plugin provided by MicroCal/GE Healthcare.
8
Circular Dichroism Analysis of Protein Thermal Stability
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CD spectra were recorded using a Jasco J-1500 spectropolarimeter (Oklahoma City, OK, USA) equipped with a Peltier element for temperature control and a six-position cuvette holder. Proteins were dialyzed in 5 mM MOPS pH:7.5; 5 mM NaCl, for 15 h, at 4 °C; 3 changes; constant stirring. Aggregated material was removed by centrifugation (20,000 g; 15 min; 4 °C) before protein concentration was determined on a Nanodrop instrument (280 nm; 2000 series; Thermo). The molecular extinction coefficient and molecular weight for A280 analysis was determined using the Expasy server (http://web.expasy.org/protparam/ ). Variable temperature measurements (15–85 °C) were performed using 15 µM protein, in 5 mM MOPS pH 7.5, 5 mM NaCl, 0.5%DMSO, in the presence or not of BDA-366 (50 µM) or ABT-737 (5 µM) (as indicated); 1 mm quartz cuvettes (Hellma, Mullheim, Germany); interval 0.5 °C; gradient 1 °C/min; DIT: 0.5 s; bandwidth: 1 nm. Data were analyzed using the SPECTRA ANALYSIS v.2 software (Jasco). Tmapp were derived by acquiring the first derivatives of the melting curves, using the calculus function of ORIGIN7 (GE).
9
Binding of HIV-1 Protease Inhibitors to hDdi2 RVP
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The ability of hDdi2 RVP to bind HIV-1 protease inhibitors was analyzed at 25 °C using a high-throughput screening Auto-iTC200 system (MicroCal, GE Healthcare Life Sciences). Aliquots (2 μl) of 120 μM protease inhibitors (saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, darunavir, GS-8374, atazanavir, brecanavir, and acetyl-pepstatin) were injected stepwise into a sample cell containing 200 μl of 10 μM hDdi2 RVP (concentration calculated based on the molecular weight of the dimer; HPLC amino acid analysis was performed). The titrations were monitored by MicroCal software implemented in Origin 7.0 (MicroCal, GE Healthcare Life Sciences).
10
Calorimetry Profiling of Glycan Binding
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The binding of mannose, chitobiose and pNP-GlcNAc (Sigma-Aldrich), GlcNAc (GLYCON Biotech, Luckenwalde, DE) and the LELTE peptide to NKR-P1A and CD69 were monitored using a VP-ITC calorimeter (MicroCal Inc., GE Healthcare, Pittsbugh, USA) at 25 °C. Typically, 10 μL aliquots of 100 μM saccharide were injected stepwise into a sample cell containing 1.43 mL of 8 μM protein solution until saturation was achieved. All the experiments were accompanied by the corresponding control experiments where the putative ligands were injected into buffer alone. The signals from the titrations were analyzed using the software Origin 7.0 (GE Healthcare, Pittsbugh, USA developed by MicroCal. Microcalorimetry titration with Concanavalin A (Sigma-Aldrich, Prague, Czech Republic) was used as a positive control and mannose as a negative control.
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