Isothermal calorimetry (ITC) studies were performed on a nano-ITC instrument (TA Instruments). In order to facilitate pure substrate binding measurements and to avoid the reaction generated by HMG CoA reduction to mevalonate, the thermodynamic properties of HMGCR binding with pravastatin were analyzed (38 (link)). HMG CoA reductase WT and mutant protein at a concentration of 20 µM in Buffer B supplemented with 1 mM NADPH in the reaction cell were injected with 50 µM pravastatin in Buffer B supplemented with 1 mM NADPH using the multiple injection mode for a total 20 7-µL injections. Heat data were plotted using Nano Analyze software™.
Protocol detail
Thermodynamics of HMGCR-Pravastatin Binding
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Other organizations : Ben-Gurion University of the Negev
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Variable analysis
independent variables
- HMGCR protein (WT and mutant)
- Thermodynamic properties of HMGCR binding with pravastatin
- Concentration of HMGCR protein (20 µM)
- Buffer B supplemented with 1 mM NADPH
- Concentration of pravastatin (50 µM)
- Multiple injection mode for a total 20 7-µL injections
- Positive control: HMGCR WT protein
- Negative control: HMGCR mutant protein
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