Тип публикации: статья из журнала

Год издания: 2010

Идентификатор DOI: 10.1074/jbc.M110.133843

Аннотация: Forster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca2+-regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Показать полностьюClytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously 15N,13C,2H-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain 1H-15N HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (KD = 0.9 mM). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Журнал: Journal of Biological Chemistry

Выпуск журнала: Т. 285, № 52

Номера страниц: 40891-40900

ISSN журнала: 00219258

Издатель: American Society for Biochemistry and Molecular Biology Inc.

• Titushin M.S. (Laboratory of Photobiology,Institute of Biophysics Russian Academy of Sciences,Siberian Branch)
• Wang B.C. (Department of Biochemistry and Molecular Biology,University of Georgia)
• Lee J. (Department of Biochemistry and Molecular Biology,University of Georgia)
• Feng Y. (National Laboratory of Biomacromolecules,Institute of Biophysics Chinese Academy of Sciences)
• Li Y. (National Laboratory of Biomacromolecules,Institute of Biophysics Chinese Academy of Sciences)
• Wang J. (National Laboratory of Biomacromolecules,Institute of Biophysics Chinese Academy of Sciences)
• Liu Z.J. (National Laboratory of Biomacromolecules,Institute of Biophysics Chinese Academy of Sciences)
• Stepanyuk G.A. (Department of Biochemistry and Molecular Biology,University of Georgia)
• Markova S.V. (Laboratory of Photobiology,Institute of Biophysics Russian Academy of Sciences,Siberian Branch)
• Vysotski E.S. (Laboratory of Photobiology,Institute of Biophysics Russian Academy of Sciences,Siberian Branch)
• Golz S. (Bayer Schering Pharma AG,BSP-GDD-GTR-TD-GT)

• Ядро РИНЦ (eLIBRARY.RU)