Advertisement

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

  • Misaki Kinoshita
  • Ju Yaen Kim
  • Yuxi Lin
  • Natalia Markova
  • Toshiharu Hase
  • Young-Ho Lee
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1498)

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.

Key words

Electron transfer Enzymatic activity Ferredoxin Ferredoxin-NADP+ reductase Isothermal titration calorimetry Nuclear magnetic resonance spectroscopy Redox protein Sulfite reductase 

Notes

Acknowledgments

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.

References

  1. 1.
    Wodak SJ, Janin J (2002) Protein modules and protein-protein interaction. Adv Protein Chem 61:21–23Google Scholar
  2. 2.
    Saitoh T, Ikegami T, Nakayama M, Teshima K, Akutsu H, Hase T (2006) NMR study of the electron transfer complex of plant ferredoxin and sulfite reductase: mapping the interaction sites of ferredoxin. J Biol Chem 281:10482–10488CrossRefPubMedGoogle Scholar
  3. 3.
    Kume S, Lee YH, Nakatsuji M, Teraoka Y, Yamaguchi K, Goto Y, Inui T (2014) Fine-tuned broad binding capability of human lipocalin-type prostaglandin D synthase for various small lipophilic ligands. FEBS Lett 588:962–969CrossRefPubMedGoogle Scholar
  4. 4.
    Kinoshita M, Kim JY, Kume S, Sakakibara Y, Sugiki T, Kojima C, Kurisu G, Ikegami T, Hase T, Kimata-Ariga Y, Lee YH (2015) Physicochemical nature of interfaces controlling ferredoxin NADP(+) reductase activity through its interprotein interactions with ferredoxin. Biochim Biophys Acta 1847:1200–1211CrossRefPubMedGoogle Scholar
  5. 5.
    Kim JY, Lee YH (2015) Sulfur. Springer 61:21–23Google Scholar
  6. 6.
    Kurisu G, Kusunoki M, Katoh E, Yamazaki T, Teshima K, Onda Y, Kimata-Ariga Y, Hase T (2001) Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP(+) reductase. Nat Struct Biol 8:117–121CrossRefPubMedGoogle Scholar
  7. 7.
    Nogues I, Martinez-Julvez M, Navarro JA, Hervas M, Armenteros L, de la Rosa MA, Brodie TB, Hurley JK, Tollin G, Gomez-Moreno C, Medina M (2003) Role of hydrophobic interactions in the flavodoxin mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena PCC 7119. Biochemistry 42:2036–2045CrossRefPubMedGoogle Scholar
  8. 8.
    Nogues I, Perez-Dorado I, Frago S, Bittel C, Mayhew SG, Gomez-Moreno C, Hermoso JA, Medina M, Cortez N, Carrillo N (2005) The ferredoxin-NADP(H) reductase from Rhodobacter capsulatus: molecular structure and catalytic mechanism. Biochemistry 44:11730–11740CrossRefPubMedGoogle Scholar
  9. 9.
    Lee YH, Ikegami T, Standley DM, Sakurai K, Hase T, Goto Y (2011) Binding energetics of ferredoxin-NADP+ reductase with ferredoxin and its relation to function. Chembiochem 12:2062–2070CrossRefPubMedGoogle Scholar
  10. 10.
    Nakayama M, Akashi T, Hase T (2000) Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin. J Inorg Biochem 82:27–32CrossRefPubMedGoogle Scholar
  11. 11.
    Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefPubMedGoogle Scholar
  12. 12.
    Lee W, Tonelli M, Markley JL (2015) NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31:1325–1327CrossRefPubMedGoogle Scholar
  13. 13.
    Maeda M, Lee YH, Ikegami T, Tamura K, Hoshino M, Yamazaki T, Nakayama M, Hase T, Goto Y (2005) Identification of the N- and C-terminal substrate binding segments of ferredoxin-NADP+ reductase by NMR. Biochemistry 44:10644–10653CrossRefPubMedGoogle Scholar
  14. 14.
    Lee YH, Tamura K, Maeda M, Hoshino M, Sakurai K, Takahashi S, Ikegami T, Hase T, Goto Y (2007) Cores and pH-dependent dynamics of ferredoxin-NADP+ reductase revealed by hydrogen/deuterium exchange. J Biol Chem 282:5959–5967CrossRefPubMedGoogle Scholar
  15. 15.
    Dominguez C, Boelens R, Bonvin AM (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731–1737CrossRefPubMedGoogle Scholar
  16. 16.
    Markova N, Hallen D (2004) The development of a continuous isothermal titration calorimetric method for equilibrium studies. Anal Biochem 331:77–88CrossRefPubMedGoogle Scholar
  17. 17.
    Loladze VV, Ermolenko DN, Makhatadze GI (2001) Heat capacity changes upon burial of polar and nonpolar groups in proteins. Protein Sci 10:1343–1352CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Aoki M, Ishimori K, Fukada H, Takahashi K, Morishima I (1998) Isothermal titration calorimetric studies on the associations of putidaredoxin to NADH-putidaredoxin reductase and P450cam. Biochim Biophys Acta 1384:180–188CrossRefPubMedGoogle Scholar
  19. 19.
    Bergqvist S, Williams MA, O'Brien R, Ladbury JE (2004) Heat capacity effects of water molecules and ions at a protein-DNA interface. J Mol Biol 336:829–842CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Misaki Kinoshita
    • 1
  • Ju Yaen Kim
    • 1
  • Yuxi Lin
    • 1
  • Natalia Markova
    • 1
  • Toshiharu Hase
    • 1
  • Young-Ho Lee
    • 1
  1. 1.Institute for Protein ResearchOsaka UniversitySuita, OsakaJapan

Personalised recommendations