title: Direct measurement of excitation transfer in the protein complex of bacterial luciferase hydroxyflavin and the associated yellow fluorescence proteins from Vibrio fischeri Y1.
abstract: Time-resolved fluorescence was used to directly measure the energy transfer rate constant in the protein-protein complex involved in the yellow bioluminescence of Vibrio fischeri, strain Y1. In this reaction the putative donor is the fluorescent transient intermediate, luciferase hydroxyflavin, which exhibits a major fluorescence lifetime of the bound flavin of 10 ns. On addition of the acceptor, the V. fischeri yellow fluorescence protein containing either FMN or riboflavin as ligand, a rapid decay time, 0.25 ns, becomes predominant. The same results are observed using rec-luciferase from Photobacterium leiognathi to produce the donor. Because of favorable spectral separation in this system, this rapid decay rate of 4 ns-1, can be directly equated to the energy transfer rate. This rate is ten times higher than the rate previously observed in the Photobacterium luciferase hydroxyflavin-lumazine protein, donor-acceptor system, derived from emission anisotropy measurements. This ten-times ratio is close to the ratio of spectral overlaps of the donor fluorescence with the acceptor absorption, between these two systems, so it is concluded that the topology of the protein complexes in both cases, must be very similar. Energy transfer is also monitored by the loss of steady-state fluorescence intensity at 460 nm of the donor, on addition of the acceptor protein. A fluorescence titration indicates that luciferase hydroxyflavin and the yellow protein complex with a 1:1 stoichiometry with a Kd of 0.7 microM (0 degree C). These parameters account for the bioluminescence spectral shifting effects observed in these reactions.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602 USA.
authors.forename: V N
authors.initials: VN
authors.lastname: Petushkov
authors.forename: B G
authors.initials: BG
authors.lastname: Gibson
authors.forename: J
authors.initials: J
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004735:Energy Transfer; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives / Q000378:metabolism*; D005453:Fluorescence; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008956:Models, Chemical; D008968:Molecular Conformation; D010776:Photobacterium / Q000378:metabolism; D011621:Pteridines / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D017186:Titrimetry; D014733:Vibrio / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011621:Pteridines; C054562:yellow fluorescent protein, Bacteria; C008297:lumazine; D005486:Flavin Mononucleotide; D008156:Luciferases