Spectral properties and function of two lumazine proteins from Photobacterium

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Abstract
The spectral properties are compared for 2 6,7-dimethyl-8-ribityllumazine proteins from marine bioluminescent bacteria, 1 from a psychrophile, Photobacterium phosphoreum, and the other from a thermophile, Photobacterium leiognathi. The visible spectral properties, which are the ones by which the protein performs its biological function of bioluminescence emission, are almost the same for the 2 proteins: at 2.degree. C and 50 mM Pi, pH 7, fluorescence quantum yield .vphi.F = 0.59 and 0.54, respectively; fluorescence lifetime .tau. = 14.4 and 14.8 ns, respectively; fluorescence maxima, both 475 nm; absorption maximum, 417 and 420 nm, respectively; circular dichroism minima at around 420 nm, both -41 .times. 103 deg cm2 dmol-1. The ligand binding sites therefore must provide very similar environments and arguments are presented that the bound ligand is relatively exposed to solvent. The dissociation equilibrium was studied by steady-state fluorescence polarization. The thermophilic protein binds the ligand with Kd (20.degree. C) = 0.016 .mu.M, 10 times more tightly than the other protein [Kd (20.degree. C) = 0.16 .mu.M]. The origin of the binding difference probably resides in differences in secondary structure. The tryptophan fluorescence spectra of the 2 proteins are different, but more significant is an observation of the decay of the tryptophan emission anisotropy. For the psychrophilic lumazine protein this anisotropy decays to zero in 1 ns, implying that its single tryptophan residue lies in a very floppy region of the protein. For the other protein, the anisotropy exhibits both a fast component and a slow one corresponding to rotation of the protein as a whole. This suggests that in the thermophilic protein the tryptophan region is held more rigidly. In both proteins, however, the ligand exhibits no independent mobility, as its rotational correlation time (respectively 19.5 and 17.5 ns, 2.degree. C) corresponds to the rotation of a sphere of hydrated MW .apprx. 30,000.