title: Biochemical and Biophysical Methods to Examine the Effects of Site-Directed Mutagenesis on Enzymatic Activities and Interprotein Interactions.
abstract: 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.
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan.
authors.forename: Misaki
authors.initials: M
authors.lastname: Kinoshita
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan.
authors.forename: Ju Yaen
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan.
authors.forename: Yuxi
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan.
authors.forename: Natalia
authors.initials: N
authors.lastname: Markova
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Hase
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan. mr0505@protein.osaka-u.ac.jp.
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Lee
mesh_terms: D001703:Biophysics / Q000379:methods; D002151:Calorimetry / Q000379:methods; D004579:Electron Transport / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000235:genetics*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics / Q000378:metabolism; D060066:Protein Interaction Maps / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D005288:Ferredoxins; D005287:Ferredoxin-NADP Reductase; D050862:Oxidoreductases Acting on Sulfur Group Donors
keywords: Electron transfer; Enzymatic activity; Ferredoxin; Ferredoxin-NADP+ reductase; Isothermal titration calorimetry; Nuclear magnetic resonance spectroscopy; Redox protein; Sulfite reductase