Lumazine protein is a highly fluorescent protein found in Photobacterium. The broad and long term goals of this program are to determine the three dimensional structure of lumazine protein, to characterize the interactions of this protein with various ligands, and to elucidate how this protein carries out its natural function as the emitter of bioluminescence in Photobacterium cells. The primary sequence of lumazine protein has been determined but all attempts to obtain X-ray diffraction quality crystals have failed. Therefore lumazine protein presents itself as a challenging problem in 2D-NMR structure methods being at the upper limit of mass (21 kD) feasible using this method. The primary sequence was determined by both chemical and genomic methods. Clones and probes for lumazine protein are therefore on hand thus enabling the sequences of natural variants to be quickly determined as well as single point mutations to be made. The natural ligand is 6,7-dimethyl-8-ribityllumazine and it is highly fluorescent both on and off the protein. It is non-covalently bound and may be replaced with other ligands satisfying certain steric requirements at the 8substituted position. This enables many spectral variants to be produced that can be studied from the point of view of ligand binding and bioluminescence properties. Major use can be made of a Picosecond Fluorescence system which is an NIH-University facility. It uses a sync-pumped Nd/YAG and dye laser, cavity dumped, as a 15-ps pulse excitation source. Lumazine protein-luciferase and lumazine protein-ligand interactions can readily determined by developed methods of fluorescence and anisotropy decay. Studies of energy transfer in the luciferase-lumazine protein complex are of particular relevance to the mechanism of bioluminescence excitation of lumazine protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028139-15
Application #
2175098
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1979-12-01
Project End
1996-01-31
Budget Start
1994-02-01
Budget End
1996-01-31
Support Year
15
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Georgia
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Athens
State
GA
Country
United States
Zip Code
30602
Petushkov, V N; Ketelaars, M; Gibson, B G et al. (1996) Interaction of Photobacterium leiognathi and Vibrio fischeri Y1 luciferases with fluorescent (antenna) proteins: bioluminescence effects of the aliphatic additive. Biochemistry 35:12086-93
Petushkov, V N; Gibson, B G; Lee, J (1996) Direct measurement of excitation transfer in the protein complex of bacterial luciferase hydroxyflavin and the associated yellow fluorescence proteins from Vibrio fischeri Y1. Biochemistry 35:8413-8
Petushkov, V N; Gibson, B G; Lee, J (1995) Properties of recombinant fluorescent proteins from Photobacterium leiognathi and their interaction with luciferase intermediates. Biochemistry 34:3300-9
Illarionov, B; Illarionova, V; Lee, J et al. (1994) Expression and properties of the recombinant lumazine (riboflavin) protein from Photobacterium leiognathi. Biochim Biophys Acta 1201:251-8
Moore, S A; James, M N; O'Kane, D J et al. (1993) Crystal structure of a flavoprotein related to the subunits of bacterial luciferase. EMBO J 12:1767-74
Lee, J (1993) Lumazine protein and the excitation mechanism in bacterial bioluminescence. Biophys Chem 48:149-58
Lee, J; Gibson, B G; O'Kane, D J et al. (1992) Fluorescence study of the ligand stereospecificity for binding to lumazine protein. Eur J Biochem 210:711-9
O'Kane, D J; Prasher, D C (1992) Evolutionary origins of bacterial bioluminescence. Mol Microbiol 6:443-9
Lee, J; Wang, Y Y; Gibson, B G (1991) Electronic excitation transfer in the complex of lumazine protein with bacterial bioluminescence intermediates. Biochemistry 30:6825-35
O'Kane, D J; Woodward, B; Lee, J et al. (1991) Borrowed proteins in bacterial bioluminescence. Proc Natl Acad Sci U S A 88:1100-4

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