Biochemical and Biophysical Methods to Examine the Effects of Site-Directed Mutagenesis on Enzymatic Activities and Interprotein Interactions

Mutations in proteins often affect interactions with partner molecules, sequentially changing their activities and functions. In order to examine mutagenic effects, we herein describe practical and detailed protocols for enzymatic activity assays using ferredoxin (Fd)-NADP+ reductase (FNR) and sulfite reductase (SiR), which are electron-transferring enzymes for the Calvin cycle and sulfur assimilation in various organisms, respectively. Methods for isothermal titration calorimetry and nuclear magnetic resonance spectroscopy, which are very useful thermodynamically and mechanically for investigating the effects of mutations on intermolecular interactions, are also described with practical examples of the Fd-FNR binding system.