Properties of recombinant fluorescent proteins from Photobacterium leiognathi and their interaction with luciferase intermediates.
Ligand binding and luciferase interaction properties of the recombinant protein corresponding to the lumazine protein gene (EMBL X56534) of Photobacterium leiognathi have been determined by fluorescence dynamics, circular dichroism, gel filtration, and SDS-PAGE. Scatchard analysis of a fluorescence titration shows that the apoprotein possess one binding site, and at 30 degrees C the KdS (microM) are as follows: 6,7-dimethyl-8-ribityllumazine, 0.26; riboflavin, 0.53; and much more weakly bound FMN, 30. All holoproteins are highly fluorescent and have absorption spectra distinct from each other and from the free ligands. The longest wavelength absorption maxima are, respectively (nm, 2 degrees C), 420, 463, and 458. Ligand binding produces no change in the far-UV circular dichroism; all have mean residual ellipticity at 210 nm of -6500 deg cm2 dmol-1, the same as the native protein. However, in the bioluminescence reaction only the lumazine holoprotein shows a bioluminescence effect. Fluorescence emission anisotropy decay was used to establish that none of these holoproteins complexed with native luciferase and that the lumazine protein alone formed a 1:1 complex with the luciferase hydroxyflavin fluorescent transient and the luciferase peroxyflavin intermediates, revealed by a dominant channel of anisotropy loss, with rotational correlation time of 2.5 ns, and attributed to excitation transfer from the luciferase flavin donor to the acceptor, the lumazine ligand. The complex stability was sufficient to allow its isolation by FPLC gel filtration and verification by SDS-PAGE. These methods also confirmed the absence of interaction of the holoflavoproteins.
Petushkov
V N
VN
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602.
Gibson
B G
BG
Lee
J
J
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005454:Fluorescence Polarization; D005798:Genes, Bacterial; D007700:Kinetics; D008024:Ligands; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D008164:Luminescent Proteins; D008969:Molecular Sequence Data; D010776:Photobacterium / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008024:Ligands; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C026648:lumazine protein, Photobacterium; D005486:Flavin Mononucleotide; D008156:Luciferases