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.
Key wordsElectron transfer Enzymatic activity Ferredoxin Ferredoxin-NADP+ reductase Isothermal titration calorimetry Nuclear magnetic resonance spectroscopy Redox protein Sulfite reductase
We thank Prof. Takahisa Ikegami (Yokohama City University, Japan), Prof. Chojiro Kojima (Osaka University, Japan), and Prof. Toshihiko Sugiki (Osaka University, Japan) for their valuable comments on NMR spectroscopy, Dr. Satoshi Kume for the HADDOCK simulation (RIKEN, Japan), and Prof. Yuji Goto for ITC (Osaka University, Japan). Y.-H.L. is supported by a Grant-in-Aid for Young Scientists (B) (15K18518 and 25870407). T.S., J.Y.K., and Y.L. received financial support from a Grant-in-Aid for Scientific Research (B) (24370021) and the Japan Society for the Promotion of Science (13J03956) and (14J04433), respectively. M.K. and J.Y.K. contributed equally to this work.
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