Workflow for characterization of enzymes under different reaction conditions

Work of the Gygli group...

Investigation 1: 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 ...

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

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

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

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

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%).

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).

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.

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.

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

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

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

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

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

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

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 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 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 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 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 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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.

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.

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.

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

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

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 ...

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 ...

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

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

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

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 ...

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

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

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