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

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

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 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 NADPH to Gre2p in 0.1% Tween-20 - KPi buffer (100 mM, pH 7.5) at 25 °C. *.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 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 apj file, the data for the analysis of the first injection peak is stored. *.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 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 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 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 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 HEPES buffer (100 mM, pH 7.5) at 25 °C. *.apj file

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 (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 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 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 NADP+ to Gre2p in HEPES 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 NADP+ to Gre2p in KPi 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 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 KPi 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. *.apj file, experiment titrating 100 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 1 mM NDK.

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. *.apj file, experiment titrating 1 mM HK.

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 PBS buffer (1x, 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 NADPH 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 HEPES buffer (100 mM, pH 7.5) at 25 °C. *.apj file

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

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)

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 (recurrent) single injections experiment with a MicroCal PEAQ ITC by Malvern.

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

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