Assays

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).

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

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%).

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

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) 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.

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.