Site-Directed Mutagenesis of Reaction Centers from Rhodobacter sphaeroides

  • M. L. Paddock
  • S. H. Rongey
  • J. W. Farchaus
  • G. Feher
  • M. Y. Okamura

Abstract

Site-directed mutagenesis is a powerful tool for studying the function of proteins. In this study we describe a genetic system for performing site-directed mutagenesis on Rhodobacter (Rb.) sphaeroides reaction centers (RCs). The RC of Rb. sphaeroides has been extensively characterized (for reviews see ref. 1 and 2) and methodologies for altering its structure have been developed (e.g. exchanging cofactors, dissociating subunits). Its three-dimensional structure has been determined by X-ray crystallography (3). Three-dimensional structural information on mutant RCs is important to distinguish between a direct effect of an altered residue and an indirect effect resulting from structural changes. A large number of RC mutants have been constructed in Rb. capsulatus, which has a well developed genetic system (4). However, at present the three-dimensional structure of the Rb. capsulatus RC has not been determined. We have used the system described here to study the mechanism of proton transfer to the reduced secondary quinone, Q B 2− , by replacing Glu-L212 with Gln and Asp. The modified characteristics of these mutant RCs are described elsewhere (5,6). Other mutations have been constructed in Rb. sphaeroides using similar systems (7,8,9).

Keywords

Proton Transfer Deletion Strain Secondary Quinone Photosynthetic Growth Broad Host Range Plasmid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    Feher, G. and Okamaura, M. Y. (1978) in The Photo synthetic Bacteria, eds. Clayton, R. K. and Sistrom, W. R. (Plenum Press, New York), pp. 349–386.Google Scholar
  2. 2.
    Breton, J. and Vermeglio, A., eds. (1988) The Photosynthetic Bacterial Reaction Center, Structure and Dynamics (Plenum Press, New York).Google Scholar
  3. 3.
    Allen, J. P., Feher, G., Yeates, T. O., Komiya, H. and Rees, D. C. (1988) Proc. Natl Acad. Sci. USA 85, 8487–8491.PubMedCrossRefGoogle Scholar
  4. 4.
    Youvan, D. C, Ismail, S. and Bylina, E. J. (1985) Gene 38, 19–30.PubMedCrossRefGoogle Scholar
  5. 5.
    Paddock, M. L., Rongey, S. H., Feher, G. and Okamura, M. Y. (1989) Proc. Natl. Acad. Sci. USA 86, in press.Google Scholar
  6. 6.
    Feher, G., McPherson, P., Paddock, M. L., Rongey, S., Schönfeld, M. and Okamura, M., these proceedings.Google Scholar
  7. 7.
    Gaul, D., Brasher, B., Martin, K. and Schenck, C. (1989) Biophys. J. 55, 181a.Google Scholar
  8. 8.
    Takahashi, E. and Wraight, C. A. (1989) Biophys. J. 55, 428a.Google Scholar
  9. 9.
    Farchaus, J. W. and Oesterhelt, D. (1989) EMBO J. 8, 47–54.PubMedGoogle Scholar
  10. 10.
    Nakamaye, K. L. and Eckstein, F. (1986) Nucleic Acids Res. 24, 9679–9698.CrossRefGoogle Scholar
  11. 11.
    Vieira, J. and Messing, J. (1987) Methods Enzymol. 153, 3–11.PubMedCrossRefGoogle Scholar
  12. 12.
    Paddock, M. L., Rongey, S. H., Abresch, E. C, Feher, G. and Okamura, M. Y. (1988) Photosynth. Res. 17, 75–96.CrossRefGoogle Scholar
  13. 13.
    Mural, R. J. (1987) Nucleic Acids R. 15, 9085.CrossRefGoogle Scholar
  14. 14.
    Vieira, J. and Messing, J. (1982) Gene 19, 259–268.PubMedCrossRefGoogle Scholar
  15. 15.
    Simon, R., Priefer, U. and Puhler, A. (1983) Biol. Technology 1, 784–791.CrossRefGoogle Scholar
  16. 16.
    Ditta, G., Schmidhauser, T., Yakobsen, E., Lu, P., Liang, X. W., Finlay, D. R., Guiney, D. and Helinski, D. R. (1985) Plasmid 13, 149–153.PubMedCrossRefGoogle Scholar
  17. 17.
    Williams, J. C, Steiner, L. A., Feher, G. and Simon, M. I. (1984) Proc. Natl. Acad. Sci. USA 81, 7303–7307.PubMedCrossRefGoogle Scholar
  18. 18.
    Sistrom, W. R. (1977) J. Bacteriol. 131, 526–532.PubMedGoogle Scholar
  19. 19.
    Allen, J. P., Lous, E. J., Feher, G., Chirino, A., Komiya, H. and Rees, D. C, these proceedings.Google Scholar

Copyright information

© Springer Science+Business Media New York 1990

Authors and Affiliations

  • M. L. Paddock
    • 1
  • S. H. Rongey
    • 1
  • J. W. Farchaus
    • 2
  • G. Feher
    • 1
  • M. Y. Okamura
    • 1
  1. 1.University of CaliforniaLa JollaUSA
  2. 2.Max-Planck Institute Fur BiochemieMartinsriedGermany

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