title: Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.
abstract: The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.
authors.affiliation: Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino 142290, Russia.
authors.forename: Andrey Yu
authors.initials: AY
authors.lastname: Gorokhovatsky
authors.forename: Victor V
authors.initials: VV
authors.lastname: Marchenkov
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Rudenko
authors.forename: Tanya V
authors.initials: TV
authors.lastname: Ivashina
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Ksenzenko
authors.forename: Nils
authors.initials: N
authors.lastname: Burkhardt
authors.forename: Gennady V
authors.initials: GV
authors.lastname: Semisotnov
authors.forename: Leonid M
authors.initials: LM
authors.lastname: Vinokurov
authors.forename: Yuli B
authors.initials: YB
authors.lastname: Alakhov
mesh_terms: D000331:Aequorin / Q000737:chemistry* / Q000528:radiation effects; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D004735:Energy Transfer; D037402:Hydrozoa / Q000378:metabolism / Q000528:radiation effects; D007700:Kinetics; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000737:chemistry* / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000528:radiation effects; D037401:Scyphozoa / Q000378:metabolism / Q000528:radiation effects
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C010497:obelin; D000331:Aequorin; D002118:Calcium