ITC binding experiments

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 ...

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 ...

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.

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.

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

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

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.

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.

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

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.

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.

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

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

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

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

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

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

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.

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

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).