PROPERTIES OF RECOMBINANT FLUORESCENT PROTEINS FROM PHOTOBACTERIUM-LEIOGNATHI AND THEIR INTERACTION WITH LUCIFERASE INTERMEDIATES

Ligand binding and luciferase interaction properties of the recombinan t protein corresponding to the lumazine protein gene (EMBL X56534) of Photobacterium leiognathi have been determined by fluorescence dynamic s, circular dichroism, gel filtration, and SDS-PAGE. Scatchard analysi s of a fluorescence titration shows that the apoprotein possess one bi nding site, and at 30 degrees C the K(d)s (mu M) are as follows: 6,7-d imethyl-8-ribityllumazine, 0.26; riboflavin, 0.53; and much more weakl y bound FMN, 30. All holoproteins are highly fluorescent and have abso rption 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-U V circular dichroism; all have mean residual ellipticity at 210 nm of -6500 deg cm(2) dmol(-1), the same as the native protein. However, in the bioluminescence reaction only the lumazine holoprotein shows a bio luminescence effect. Fluorescence emission anisotropy decay was used t o establish that none of these holoproteins complexed with native luci ferase and that the lumazine protein alone formed a 1:1 complex with t he luciferase hydroxyflavin fluorescent transient and the luciferase p eroxyflavin intermediates, revealed by a dominant channel of anisotrop y 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 holoflavoprot eins.