Abstract

The mechanism by which Lumazine Protein functions in the bioluminescence reaction with bacterial luciferase will be studied using fluorescence spectra, lifetime and anisotropy decay techniques. The natural ligand, 6,7-dimethyl-8-ribityllumazine can be replaced with analogs to determine the factors responsible for binding and function of this protein. Major use will be made of a Picosecond Fluorescence Lifetime system for this research. This is an NIH-University facility and uses a sync-pumped dye laser as and excitation source. Modifications of the protein structure will also be introduced to see what effects these have on both fluorescence and bioluminescence spectral properties. Modifications will be by using natural variants of lumazine protein whose primary sequence will be needed to be determined, by derivitizing certain amino acid residues, and by site-specific mutation, if the lumazine protein is successfully cloned. This cloning project is already underway in collaboration with another group. The effect of these modifications on certain of the protein properties will also be studied. Crystallization of lumazine proteins and their complexes with luciferases will also be done. This will enable some preliminary 3-dimensional structural information to be measured and collaboration with a fully equipped X-ray structure group then to be developed.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028139-11
Application #
3275396
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1979-12-01
Project End
1991-01-31
Budget Start
1990-02-01
Budget End
1991-01-31
Support Year
11
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
University of Georgia
Department
Type
Schools of Arts and Sciences
DUNS #
City
Athens
State
GA
Country
United States
Zip Code
30602

  Publications

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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