Workflow for characterization of enzymes under different reaction conditions

An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are dynamic light scattering (DLS), UV-Vis spectrophotometry and isothermal titration calorimetry (ITC) respectively. The workflow is standardized by the use of SOPs and python-scripted data analysis. We have used the NADPH-dependent alcohol dehydrogenase Gre2p as a challenging enzyme to demonstrate the power of this workflow. Our work highlights the utility for combined binding and kinetic studies for such complex multi-substrate reactions and the importance of sample quality control during experiments.

DOI: 10.15490/fairdomhub.1.investigation.464.1

Zenodo URL: None

Created at: 20th Jul 2021 at 09:27

Contents

Spectrophotometric Activity Measurements

Measure Gre2p activity by following the change in NADPH absorbance at 340 nm for the conversion of different substrates.

Specific activity of Gre2p

Specific activity of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK).

Specific activity of Gre2p after different treatments (.opju)

Specific activity of Gre2p was measured after different treatments (none, on ice, at room temperature, in the ITC, stirred, shaken). These data are used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs.
This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

  • activity_Gre2p(f_ITC_RT_5hice)_25_100_250nM_NDK_NADPH_absorbance_340nm.opju

Specific activity of Gre2p after different treatments (.xlsx)

Specific activity of Gre2p was measured after different treatments (none, on ice, at room temperature, in the ITC, stirred, shaken). These data are used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs.
These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

  • activity_Gre2p(f_ITC_RT_5hice)_25_100_250nM_NDK_NADPH_absorbance_340nm.xpt.xlsx

Specific activity of Gre2p after incubations with different Tween concentrations (.opju)

Specific activity of Gre2p was measured after incubation with different Tween-20 concentrations (none, 0.01, 0.1, 1.0%). These data are also used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs.
This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

  • activity_Gre2p_REPETITON(tween20_0_0,1%_1%)_25_100_250nM_NDK_NADPH_absorbance_340nm.opju

Specific activity of Gre2p after incubations with different Tween concentrations (.xlsx)

Specific activity of Gre2p was measured after incubation with different Tween-20 concentrations (none, 0.01, 0.1, 1.0%). These data are also used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs.
These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

  • activity_Gre2p_REPETITION(tween20_0_0,1%_1%)_25_100_250nM_NDK_NADPH_absorbance_340nm.xlsx

SOP for specific activity measurements

SOP describing how to perform specific activity measurements with Gre2p in multiwell plates (96w) using an UV-Vis Plate reader (Synergy H1).

  • SOP_UVVIS#2_SpAc_Gre2p.pdf

Python Workflow for Analysis of Specific Activities and DLS data

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween.
Also analysis of DLS data of homogeneity of Gre2p samples measured before and after these treatments.

A python workflow is used to analyse the data and create a plot of the data.
It requires the following directory structure:

./Script_for_S13andS18.py
./Script_for_S22.py
./Script_for_S15.py

and as
...

  • python_DLSandSpAc_1.1.zip

Kinetic parameters of Gre2p

Kinetic parameters (Km, kcat) of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK) or hexane-2,5-dione.
Initial rates at different substrate concentrations are measured.

Enzyme kinetics measurement of Gre2p at different substrate (NDK) concentrations (.opju)

Activity (initial rates) of Gre2p were measured followng NADPH absorbance at different substrate [NDK] concentrations. Initial rates are calculated (mM/s) and plotted as a function of [NDK] to allow fitting with a enzyme kinetic model, e.g. Michaelis Menten.
This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

  • MM_25nM_Gre2p_NDK_NADPH_absorbance_340nm.opju

Enzyme kinetics measurement of Gre2p at different substrate (NDK) concentrations (.xlsx)

Activity (initial rates) of Gre2p were measured followng NADPH absorbance at different substrate [NDK] concentrations. Initial rates are calculated (mM/s) and plotted as a function of [NDK] to allow fitting with a enzyme kinetic model, e.g. Michaelis Menten.
These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

  • MM_25nM_Gre2p_NDK_NADPH_absorbance_340nm.xlsx

Enzyme kinetics measurement of Gre2p at different substrate (hexane-2,5-dione) concentrations (.opju)

Activity (initial rates) of Gre2p were measured followng NADPH absorbance at different substrate [hexane-2,5-dione] concentrations. Initial rates are calculated (mM/s) and plotted as a function of [hexane-2,5-dione] to allow fitting with a enzyme kinetic model, e.g. Michaelis Menten.
This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

  • MM-SI_10nM_Gre2p_Hexane-2,5-dione_NADPH_absorbance_340nm.opju

Enzyme kinetics measurement of Gre2p at different substrate (hexane-2,5-dione) concentrations (.xlsx)

Activity (initial rates) of Gre2p were measured followng NADPH absorbance at different substrate [hexane-2,5-dione] concentrations. Initial rates are calculated (mM/s) and plotted as a function of [hexane-2,5-dione] to allow fitting with a enzyme kinetic model, e.g. Michaelis Menten.
These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

  • MM-SI_10nM_Gre2p_Hexane-2,5-dione_NADPH_absorbance_340nm.xlsx

SOP for enzyme kinetics measurements

SOP describing how to perform activity measurements (inital rates) with Gre2p in multiwell plates (96w) using an UV-Vis Plate reader (Synergy H1).

  • SOP_UVVIS#1_EnzKin_Gre2p.pdf

Selwyn test of Gre2p

Specific activity of Gre2p measured by following the change in absorbance of NADPH at 340 nm for the conversion of nitrononane-2,8-dione (NDK) using different enzyme concentrations.

Specific activity of Gre2p after different treatments (.opju)

Specific activity of Gre2p was measured after different treatments (none, on ice, at room temperature, in the ITC, stirred, shaken). These data are used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs.
This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

  • activity_Gre2p(f_ITC_RT_5hice)_25_100_250nM_NDK_NADPH_absorbance_340nm.opju

Specific activity of Gre2p after different treatments (.xlsx)

Specific activity of Gre2p was measured after different treatments (none, on ice, at room temperature, in the ITC, stirred, shaken). These data are used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs.
These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

  • activity_Gre2p(f_ITC_RT_5hice)_25_100_250nM_NDK_NADPH_absorbance_340nm.xpt.xlsx

Specific activity of Gre2p after incubations with different Tween concentrations (.opju)

Specific activity of Gre2p was measured after incubation with different Tween-20 concentrations (none, 0.01, 0.1, 1.0%). These data are also used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs.
This is a file that can be opened with the Origin Software for analysis and contains data copied in manually from the xlsx.

  • activity_Gre2p_REPETITON(tween20_0_0,1%_1%)_25_100_250nM_NDK_NADPH_absorbance_340nm.opju

Specific activity of Gre2p after incubations with different Tween concentrations (.xlsx)

Specific activity of Gre2p was measured after incubation with different Tween-20 concentrations (none, 0.01, 0.1, 1.0%). These data are also used to perform a Selwyn Test to determine if enzyme inactivation or slow-onset inhibition occurs.
These are the raw data obtained directly from the Synergy UV-Vis Spectrophotometer.

  • activity_Gre2p_REPETITION(tween20_0_0,1%_1%)_25_100_250nM_NDK_NADPH_absorbance_340nm.xlsx

SOP for specific activity measurements

SOP describing how to perform specific activity measurements with Gre2p in multiwell plates (96w) using an UV-Vis Plate reader (Synergy H1).

  • SOP_UVVIS#2_SpAc_Gre2p.pdf

Python Workflow for Selwyn test

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween.

A python workflow is used to analyse the data and create a plot where the outcomes of the Selwyn test are plotted.
It requires the following directory structure:

./Sewlyn_test_forS19.py
./tween/M1.csv
./tween/M2.csv
./tween/M3.csv
./tween/M4.csv
./treatments/M1.csv
./treatments/M2.csv
./treatments/M3.csv
...

  • python_Selwyntest.zip

ITC binding experiments

Measure binding of reactants to Gre2p by ITC (isothermal titration calorimetry).

Binding of NADPH to Gre2p in KPi Buffer (ITC-BIND)

ITC binding (BIND) experiment to determine the binding parameters of NADPH to Gre2p in 100 mM KPi Buffer.

ITC-BIND experiment for NADPH binding to Gre2p in KPi buffer (.apj)

ITC binding experiment for the binding of NADPH to Gre2p in KPi buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • KPI_NADPH.apj

ITC-BIND experiment for NADPH binding to Gre2p in KPi buffer (.csv)

ITC binding experiment for the binding of NADPH to Gre2p in KPi buffer (100 mM, pH 7.5) at 25 °C.
*.csv file.

  • KPI_NADPH.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

Binding of NADPH to Gre2p in PBS Buffer (ITC-BIND)

ITC binding (BIND) experiment to determine the binding parameters of NADPH to Gre2p in 1x PBS Buffer.

ITC-BIND experiment for NADPH binding to Gre2p in PBS buffer (.apj)

ITC binding experiment for the binding of NADPH to Gre2p in PBS buffer (1x, pH 7.5) at 25 °C.
*.apj file

  • PBS_NADPH.apj

ITC-BIND experiment for NADPH binding to Gre2p in PBS buffer (.csv)

ITC binding experiment for the binding of NADPH to Gre2p in PBS buffer (1x, pH 7.5) at 25 °C.
*.csv file.

  • PBS_NADPH.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

Binding of NADPH to Gre2p in HEPES Buffer (ITC-BIND)

ITC binding (BIND) experiments to determine the binding parameters of NADPH to Gre2p in 100 mM HEPES Buffer.

ITC-BIND experiment for NADPH binding to Gre2p in HEPES buffer (.apj)

ITC binding experiment for the binding of NADPH to Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • HEPES_NADPH.apj

ITC-BIND experiment for NADPH binding to Gre2p in HEPES buffer (.csv)

ITC binding experiment for the binding of NADPH to Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.csv file.

  • HEPES_NADPH.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

Binding of NADP+ to Gre2p in KPi Buffer (ITC-BIND)

ITC binding (BIND) experiments to determine the binding parameters of NADP+ to Gre2p in 100 mM KPi Buffer.

ITC-BIND experiment for NADP+ binding to Gre2p in KPi buffer (.apj)

ITC binding experiment for the binding of NADP+ to Gre2p in KPi buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • KPI_NADPplus.apj

ITC-BIND experiment for NADP+ binding to Gre2p in KPi buffer (.csv)

ITC binding experiment for the binding of NADP+ to Gre2p in KPi buffer (100 mM, pH 7.5) at 25 °C.
*.csv file.

  • KPI_NADPplus.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

Binding of NADP+ to Gre2p in PBS Buffer (ITC-BIND)

ITC binding (BIND) experiments to determine the binding parameters of NADP+ to Gre2p in 1x PBS Buffer.

ITC-BIND experiment for NADP+ binding to Gre2p in PBS buffer (.apj)

ITC binding experiment for the binding of NADP+ to Gre2p in PBS buffer (1x, pH 7.5) at 25 °C. Note that for successfull fitting, the stoichiometry of binding had to be fixed at 0.85, in agreement with data from the other binding experiments.
*.apj file

  • PBS_NADPplus_85.apj

ITC-BIND experiment for NADP+ binding to Gre2p in PBS buffer (.csv)

ITC binding experiment for the binding of NADP+ to Gre2p in PBS buffer (1x, pH 7.5) at 25 °C. Note that for successfull fitting, the stoichiometry of binding had to be fixed at 0.85, in agreement with data from the other binding experiments.
*.csv file.

  • PBS_NADPplus_85.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

Binding of NADP+ to Gre2p in HEPES Buffer (ITC-BIND)

ITC binding (BIND) experiments to determine the binding parameters of NADP+ to Gre2p in 100 mM HEPES Buffer.

ITC-BIND experiment for NADP+ binding to Gre2p in HEPES buffer (.apj)

ITC binding experiment for the binding of NADP+ to Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • HEPES_NADPplus_filtered.apj

ITC-BIND experiment for NADP+ binding to Gre2p in HEPES buffer (.csv)

ITC binding experiment for the binding of NADP+ to Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.csv file.

  • HEPES_NADPplus_filtered.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

Binding of NDK to Gre2p (ITC-BIND)

ITC binding (BIND) experiments to determine the binding parameters of NDK (nitrononane-2,8-dione) to Gre2p in 100 mM KPi Buffer.
Experiments failed due to very weak binding and poor solubility of NDK in buffer.

ITC-BIND experiment for NDK binding to Gre2p (.apj)

ITC binding experiment for the binding of nitrononane-2,8-dione (NDK) to Gre2p. Note that binding was unsuccessful.
*.apj file, experiment titrating 1 mM NDK.

  • NDK_failed_binding_1mM.apj

ITC-BIND experiment for NDK binding to Gre2p (.apj)

ITC binding experiment for the binding of nitrononane-2,8-dione (NDK) to Gre2p. Note that binding was unsuccessful.
*.apj file, experiment titrating 100 mM NDK.

  • NDK_failed_binding_100mM.apj

ITC-BIND experiment for NDK binding to Gre2p (.csv)

ITC binding experiment for the binding of nitrononane-2,8-dione (NDK) to Gre2p. Note that binding was unsuccessful.
*.csv file, experiment titrating 1 mM NDK.

  • NDK_failed_binding_1mM.csv

ITC-BIND experiment for NDK binding to Gre2p (.csv)

ITC binding experiment for the binding of nitrononane-2,8-dione (NDK) to Gre2p. Note that binding was unsuccessful.
*.csv file, experiment titrating 100 mM NDK.

  • NDK_failed_binding_100mM.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

Binding of HK to Gre2p (ITC-BIND)

ITC binding (BIND) experiments to determine the binding parameters of HK ((5S,8S)-anti hydroxyketone) to Gre2p in 100 mM KPi Buffer.
Experiments failed due to very weak binding and poor solubility of HK in buffer.

ITC-BIND experiment for HK binding to Gre2p (.apj)

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful.
*.apj file, experiment titrating 1 mM HK.

  • HK_failed_binding_1mM.apj

ITC-BIND experiment for HK binding to Gre2p (.apj)

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful.
*.apj file, experiment titrating 200 mM HK.

  • HK_failed_binding_200mM.apj

ITC-BIND experiment for HK binding to Gre2p (.csv)

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful.
*.csvfile, experiment titrating 1 mM HK.

  • HK_failed_binding_1mM.csv

ITC-BIND experiment for HK binding to Gre2p (.csv)

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful.
*.csv file, experiment titrating 200 mM HK.

  • HK_failed_binding_200mM.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

Analysis of data from ITC experiments (binding)

Python workflow for the analysis of ITC-BIND, ITC-MIM and ITC-(r)SIM experiments. Organized in a *.zip folder.
Requires the following directory structure:

./ITC_analysis.py
./input/BINDING/*.apj
./input/BINDING/*.csv
./input/KINETICS/*.apj
./input/KINETICS/*.csv
./scripts/binding_neu.py
./scripts/kinetics_neu.py

And can be executed by running python ITC_analysis.py in the directory.
Filenames for the input *.apj and *.csv files are defined in ITC_analysis.py.
The output directory is written by
...

ITC-BIND experiment for NADPH binding to Gre2p in HEPES buffer (.apj)

ITC binding experiment for the binding of NADPH to Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • HEPES_NADPH.apj

ITC-BIND experiment for NADP+ binding to Gre2p in HEPES buffer (.apj)

ITC binding experiment for the binding of NADP+ to Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • HEPES_NADPplus_filtered.apj

ITC-BIND experiment for NADPH binding to Gre2p in KPi buffer (.apj)

ITC binding experiment for the binding of NADPH to Gre2p in KPi buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • KPI_NADPH.apj

ITC-BIND experiment for NADP+ binding to Gre2p in KPi buffer (.apj)

ITC binding experiment for the binding of NADP+ to Gre2p in KPi buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • KPI_NADPplus.apj

ITC-BIND experiment for NADPH binding to Gre2p in PBS buffer (.apj)

ITC binding experiment for the binding of NADPH to Gre2p in PBS buffer (1x, pH 7.5) at 25 °C.
*.apj file

  • PBS_NADPH.apj

ITC-BIND experiment for NADP+ binding to Gre2p in PBS buffer (.apj)

ITC binding experiment for the binding of NADP+ to Gre2p in PBS buffer (1x, pH 7.5) at 25 °C. Note that for successfull fitting, the stoichiometry of binding had to be fixed at 0.85, in agreement with data from the other binding experiments.
*.apj file

  • PBS_NADPplus_85.apj

ITC-BIND experiment for HK binding to Gre2p (.apj)

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful.
*.apj file, experiment titrating 1 mM HK.

  • HK_failed_binding_1mM.apj

ITC-BIND experiment for HK binding to Gre2p (.apj)

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful.
*.apj file, experiment titrating 200 mM HK.

  • HK_failed_binding_200mM.apj

ITC-BIND experiment for NDK binding to Gre2p (.apj)

ITC binding experiment for the binding of nitrononane-2,8-dione (NDK) to Gre2p. Note that binding was unsuccessful.
*.apj file, experiment titrating 1 mM NDK.

  • NDK_failed_binding_1mM.apj

ITC-BIND experiment for NDK binding to Gre2p (.apj)

ITC binding experiment for the binding of nitrononane-2,8-dione (NDK) to Gre2p. Note that binding was unsuccessful.
*.apj file, experiment titrating 100 mM NDK.

  • NDK_failed_binding_100mM.apj

ITC-BIND experiment for NADPH binding to Gre2p in KPi buffer (.csv)

ITC binding experiment for the binding of NADPH to Gre2p in KPi buffer (100 mM, pH 7.5) at 25 °C.
*.csv file.

  • KPI_NADPH.csv

ITC-BIND experiment for NADPH binding to Gre2p in HEPES buffer (.csv)

ITC binding experiment for the binding of NADPH to Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.csv file.

  • HEPES_NADPH.csv

ITC-BIND experiment for NADPH binding to Gre2p in PBS buffer (.csv)

ITC binding experiment for the binding of NADPH to Gre2p in PBS buffer (1x, pH 7.5) at 25 °C.
*.csv file.

  • PBS_NADPH.csv

ITC-BIND experiment for NADP+ binding to Gre2p in KPi buffer (.csv)

ITC binding experiment for the binding of NADP+ to Gre2p in KPi buffer (100 mM, pH 7.5) at 25 °C.
*.csv file.

  • KPI_NADPplus.csv

ITC-BIND experiment for NADP+ binding to Gre2p in PBS buffer (.csv)

ITC binding experiment for the binding of NADP+ to Gre2p in PBS buffer (1x, pH 7.5) at 25 °C. Note that for successfull fitting, the stoichiometry of binding had to be fixed at 0.85, in agreement with data from the other binding experiments.
*.csv file.

  • PBS_NADPplus_85.csv

ITC-BIND experiment for NADP+ binding to Gre2p in HEPES buffer (.csv)

ITC binding experiment for the binding of NADP+ to Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.csv file.

  • HEPES_NADPplus_filtered.csv

ITC-BIND experiment for NDK binding to Gre2p (.csv)

ITC binding experiment for the binding of nitrononane-2,8-dione (NDK) to Gre2p. Note that binding was unsuccessful.
*.csv file, experiment titrating 1 mM NDK.

  • NDK_failed_binding_1mM.csv

ITC-BIND experiment for NDK binding to Gre2p (.csv)

ITC binding experiment for the binding of nitrononane-2,8-dione (NDK) to Gre2p. Note that binding was unsuccessful.
*.csv file, experiment titrating 100 mM NDK.

  • NDK_failed_binding_100mM.csv

ITC-BIND experiment for HK binding to Gre2p (.csv)

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful.
*.csvfile, experiment titrating 1 mM HK.

  • HK_failed_binding_1mM.csv

ITC-BIND experiment for HK binding to Gre2p (.csv)

ITC binding experiment for the binding of (5S,8S)-anti hydroxyketone (HK) to Gre2p. Note that binding was unsuccessful.
*.csv file, experiment titrating 200 mM HK.

  • HK_failed_binding_200mM.csv

ITC-BIND experiment for NADPH binding to Gre2p in Tween buffer (.apj)

ITC binding experiment for the binding of NADPH to Gre2p in 0.1% Tween-20 - KPi buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • Tween_NADPH_filtered.apj

ITC-BIND experiment for NADPH binding to Gre2p in Tween buffer (.csv)

ITC binding experiment for the binding of NADPH to Gre2p in 0.1% Tween-20 - KPi buffer (100 mM, pH 7.5) at 25 °C.
*.csv file

  • Tween_NADPH_filtered.csv

Python Workflow for analysis of ITC data

Python workflow for the analysis of ITC-BIND, ITC-MIM and ITC-(r)SIM experiments. Organized in a *.zip folder.
Requires the following directory structure:

./ITC_analysis.py
./input/BINDING/*.apj
./input/BINDING/*.csv
./input/KINETICS/*.apj
./input/KINETICS/*.csv
./scripts/binding_neu.py
./scripts/kinetics_neu.py

And can be executed by running python ITC_analysis.py in the directory.
Filenames for the input *.apj and *.csv files are defined in ITC_analysis.py.
The output directory is written by
...

  • python_ITC.zip

Binding of NADPH to Gre2p in Tween-KPi Buffer (ITC-BIND)

ITC binding (BIND) experiment to determine the binding parameters of NADPH to Gre2p in 100 mM KPi Buffer with 0.1% Tween-20 added.

ITC-BIND experiment for NADPH binding to Gre2p in Tween buffer (.apj)

ITC binding experiment for the binding of NADPH to Gre2p in 0.1% Tween-20 - KPi buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • Tween_NADPH_filtered.apj

ITC-BIND experiment for NADPH binding to Gre2p in Tween buffer (.csv)

ITC binding experiment for the binding of NADPH to Gre2p in 0.1% Tween-20 - KPi buffer (100 mM, pH 7.5) at 25 °C.
*.csv file

  • Tween_NADPH_filtered.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-BIND experiments

SOP describing how to perform an ITC binding experiment with a MicroCal PEAQ ITC by Malvern.
Note: the DOIs from the Chemotion database for the synthesis of HK and NDK are missing in this version beacuse the DOIs do not exist yet (as of 27.04.21)

  • SOP_ITC#4_BIND_Gre2p.pdf

ITC kinetic experiments (enzyme activity)

Measure kinetics of reactants by Gre2p with ITC (isothermal titration calorimetry).

Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-MIM) in KPi buffer

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 100 mM KPi buffer pH 7.5
Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in KPi buffer (.apj)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in KPI buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • KPI_MIM_filtered.apj

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in KPi buffer (.csv)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in KPI buffer (100 mM, pH 7.5) at 25 °C.
*.csv file

  • KPI_MIM_filtered.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-MIM experiments

SOP describing how to perform an ITC multiple injections experiment with a MicroCal PEAQ ITC by Malvern.

  • SOP_ITC#2_MIM_Gre2p.pdf

Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-MIM) in PBS buffer

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 1x PBS buffer pH 7.5
Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (.apj)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (1x, pH 7.5) at 25 °C.
*.apj file

  • PBS_MIM_filtered.apj

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (.csv)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (1x, pH 7.5) at 25 °C.
*.csv file

  • PBS_MIM_filtered.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-MIM experiments

SOP describing how to perform an ITC multiple injections experiment with a MicroCal PEAQ ITC by Malvern.

  • SOP_ITC#2_MIM_Gre2p.pdf

Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-MIM) in HEPES buffer

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 100 mM HEPES buffer pH 7.5
Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (.apj)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • HEPES_MIM_filtered.apj

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (.csv)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.csv file

  • HEPES_MIM_filtered.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-MIM experiments

SOP describing how to perform an ITC multiple injections experiment with a MicroCal PEAQ ITC by Malvern.

  • SOP_ITC#2_MIM_Gre2p.pdf

Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-rSIM) in 3 buffers

ITC recurrent single injection (rSIM) to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 100 mM KPi buffer, 1x PBS buffer and 100 mM HEPES buffer, all with pH 7.5

ITC-rSIM experiment for the reaction of NADPH and NDK with Gre2p (.apj)

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5).
In this apj file, the data for the analysis of the first injection peak is stored.
*.apj file

  • buffers_SIM_1stpeak_including_controls.apj

ITC-rSIM experiment for the reaction of NADPH and NDK with Gre2p (.apj)

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5).
In this apj file, the data for the analysis of the second injection peak is stored.
*.apj file

  • buffers_SIM_2ndpeak.apj

ITC-rSIM experiment for the reaction of NADPH and NDK with Gre2p (.csv)

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5).
In this csv file, the data for the analysis of the both injection peaks is stored.
*.csv file

  • recurrent_SIM_withcontrols.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-(r)SIM experiments

SOP describing how to perform an ITC (recurrent) single injections experiment with a MicroCal PEAQ ITC by Malvern.

  • SOP_ITC#3_(r)SIM_Gre2p.pdf

Analysis of data from ITC experiments (kinetics)

Python workflow for the analysis of ITC-BIND, ITC-MIM and ITC-(r)SIM experiments. Organized in a *.zip folder.
Requires the following directory structure:

./ITC_analysis.py
./input/BINDING/*.apj
./input/BINDING/*.csv
./input/KINETICS/*.apj
./input/KINETICS/*.csv
./scripts/binding_neu.py
./scripts/kinetics_neu.py

And can be executed by running python ITC_analysis.py in the directory.
Filenames for the input *.apj and *.csv files are defined in ITC_analysis.py.
The output directory is written by
...

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (.apj)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • HEPES_MIM_filtered.apj

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (.csv)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in HEPES buffer (100 mM, pH 7.5) at 25 °C.
*.csv file

  • HEPES_MIM_filtered.csv

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in KPi buffer (.apj)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in KPI buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • KPI_MIM_filtered.apj

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in KPi buffer (.csv)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in KPI buffer (100 mM, pH 7.5) at 25 °C.
*.csv file

  • KPI_MIM_filtered.csv

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (.apj)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (1x, pH 7.5) at 25 °C.
*.apj file

  • PBS_MIM_filtered.apj

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (.csv)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in PBS buffer (1x, pH 7.5) at 25 °C.
*.csv file

  • PBS_MIM_filtered.csv

ITC-rSIM experiment for the reaction of NADPH and NDK with Gre2p (.apj)

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5).
In this apj file, the data for the analysis of the first injection peak is stored.
*.apj file

  • buffers_SIM_1stpeak_including_controls.apj

ITC-rSIM experiment for the reaction of NADPH and NDK with Gre2p (.apj)

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5).
In this apj file, the data for the analysis of the second injection peak is stored.
*.apj file

  • buffers_SIM_2ndpeak.apj

ITC-rSIM experiment for the reaction of NADPH and NDK with Gre2p (.csv)

ITC recurrent single injection experiment for the reaction of NADPH and NDK with Gre2p in KPi, PBS and HEPES buffer (100, 1x, 100 mM, respectively, all at 25°C and pH 7.5).
In this csv file, the data for the analysis of the both injection peaks is stored.
*.csv file

  • recurrent_SIM_withcontrols.csv

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in Tween-KPi buffer (.apj)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in 0.1%Tween-20-KPI buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • Tween_MIM_filtered.apj

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in Tween-KPi buffer (.csv)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in 0.1%Tween-20-KPI buffer (100 mM, pH 7.5) at 25 °C.
*.csv file

  • Tween_MIM_filtered.csv

Python Workflow for analysis of ITC data

Python workflow for the analysis of ITC-BIND, ITC-MIM and ITC-(r)SIM experiments. Organized in a *.zip folder.
Requires the following directory structure:

./ITC_analysis.py
./input/BINDING/*.apj
./input/BINDING/*.csv
./input/KINETICS/*.apj
./input/KINETICS/*.csv
./scripts/binding_neu.py
./scripts/kinetics_neu.py

And can be executed by running python ITC_analysis.py in the directory.
Filenames for the input *.apj and *.csv files are defined in ITC_analysis.py.
The output directory is written by
...

  • python_ITC.zip

Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-MIM) in Tween- KPi buffer

ITC multiple injection (MIM) experiments to determine the kinetic parameters of NDK (nitrononane-2,8-dione) and NADPH with Gre2p in 0.1%Tween-20-100 mM KPi buffer pH 7.5
Data from ITC recurrent single injection (rSIM) experiments were used to achieve proper fitting of kcat values.

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in Tween-KPi buffer (.apj)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in 0.1%Tween-20-KPI buffer (100 mM, pH 7.5) at 25 °C.
*.apj file

  • Tween_MIM_filtered.apj

ITC-MIM experiment for the reaction of NADPH and NDK with Gre2p in Tween-KPi buffer (.csv)

ITC multiple injection experiment for the reaction of NADPH and NDK with Gre2p in 0.1%Tween-20-KPI buffer (100 mM, pH 7.5) at 25 °C.
*.csv file

  • Tween_MIM_filtered.csv

SOP for usage of the ITC

SOP describing the steps for use of the ITC device (MicroCal PEAQ ITC by Malvern).

  • SOP_ITC#1_General.pdf

SOP for ITC-MIM experiments

SOP describing how to perform an ITC multiple injections experiment with a MicroCal PEAQ ITC by Malvern.

  • SOP_ITC#2_MIM_Gre2p.pdf

DLS measurements (homogeneity of an enzyme sample)

Measure homogeneity of an enzyme sample (Gre2p) with DLS (dynamic light scattering).

DLS measurements in KPi Buffer and in KPi buffer with Tween added

Homogeneity of the Gre2p samples in KPi Buffer before and after different treatments, including different amounts of tween-20 in the buffer (0.01, 0.1, 1.0%).

DLS data for Gre2p in KPi Buffer and in KPi buffer with Tween added

Raw data of the measurements obtained via DLS measurements. Can be opened with the Zetasizer software.

  • DLS_KPiandTween.dts

SOP for DLS measurements

SOP describing how to perform Dynamic Light Scattering (DLS) measurements with Gre2p in cuvettes using a Zetasizer Nano DLS reader.

  • SOP_DLS#1_ProtStab_Gre2p_1.1.pdf

Python Workflow for Analysis of Specific Activities and DLS data

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween.
Also analysis of DLS data of homogeneity of Gre2p samples measured before and after these treatments.

A python workflow is used to analyse the data and create a plot of the data.
It requires the following directory structure:

./Script_for_S13andS18.py
./Script_for_S22.py
./Script_for_S15.py

and as
...

  • python_DLSandSpAc_1.1.zip

DLS measurements in 2 buffers

Homogeneity of the Gre2p samples in HEPES and PBS Buffer before and after different treatments.

DLS data for Gre2p in HEPES and PBS buffer

Raw data of the measurements obtained via DLS measurements. Can be opened with the Zetasizer software.

  • DLS_PBSandHEPES.dts

SOP for DLS measurements

SOP describing how to perform Dynamic Light Scattering (DLS) measurements with Gre2p in cuvettes using a Zetasizer Nano DLS reader.

  • SOP_DLS#1_ProtStab_Gre2p_1.1.pdf

Python Workflow for Analysis of Specific Activities and DLS data

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween.
Also analysis of DLS data of homogeneity of Gre2p samples measured before and after these treatments.

A python workflow is used to analyse the data and create a plot of the data.
It requires the following directory structure:

./Script_for_S13andS18.py
./Script_for_S22.py
./Script_for_S15.py

and as
...

  • python_DLSandSpAc_1.1.zip

DLS measurements in KPi buffer with BSA added

Homogeneity of the Gre2p samples in KPi Buffer before and after different treatments, including different amounts of BSA in the buffer (7.5 µM and 75 µM).

DLS data for Gre2p in KPi buffer with BSA added

Raw data of the measurements obtained via DLS measurements. Can be opened with the Zetasizer software.

  • DLS_BSA.dts

SOP for DLS measurements

SOP describing how to perform Dynamic Light Scattering (DLS) measurements with Gre2p in cuvettes using a Zetasizer Nano DLS reader.

  • SOP_DLS#1_ProtStab_Gre2p_1.1.pdf

Python Workflow for Analysis of Specific Activities and DLS data

Gre2p activity monitored by NADPH absorbance using different enzyme concentrations. Activity is measured after different storage conditions (treatments) and in presence of different amounts of tween.
Also analysis of DLS data of homogeneity of Gre2p samples measured before and after these treatments.

A python workflow is used to analyse the data and create a plot of the data.
It requires the following directory structure:

./Script_for_S13andS18.py
./Script_for_S22.py
./Script_for_S15.py

and as
...

  • python_DLSandSpAc_1.1.zip
Fingerprints

These checksums allow you to check a Snapshot you have downloaded hasn't been modified. For details on how to use these please visit this guide

MD5: 53fabb99a97ae7033d14cf2d10188b6f

SHA1: a1195e0ada32069e1dc8383b09227552e118e77e

Citation
Gygli, G., & Ott, F. (2021). Workflow for characterization of enzymes under different reaction conditions. FAIRDOMHub. https://doi.org/10.15490/FAIRDOMHUB.1.INVESTIGATION.464.1
Snapshots
Snapshot 1 (20th Jul 2021) DOI
Activity

Views: 27   Downloads: 0

Created: 20th Jul 2021 at 09:27

Last updated: 20th Jul 2021 at 09:28

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