title: Fluorescence study of the ligand stereospecificity for binding to lumazine protein.
abstract: 6,7-Dimethyllumazine derivatives, substituted at the 8-position with aldityls or monohydroxyalkyl groups, have been examined for their binding ability to lumazine apo-protein from two strains of Photobacterium phosphoreum using fluorescence dynamics techniques. On the protein the lumazine has a nearly monoexponential decay of fluorescence with lifetime 13.8 ns (20 degrees C). In free solution the lifetime is 9.6 ns. The concentration of free and bound lumazine in an equilibrium mixture can be recovered readily by analysis of the fluorescence decay. Only the aldityl derivatives D-xylityl and 3'-deoxy-D-ribityl, having stereoconfigurations at the 2' and 4' positions identical to the natural ligand, 8-(1'-D-ribityl), show comparable dissociation constants (0.3 microM, 20 degrees C, pH 7.0). D-Erythrityl and L-arabityl have dissociation constants of 1-2 microM. All other ligands show no interaction at all or have dissociation constants in the range 6-80 microM, which can still be determined semi-quantitatively using the fluorescence decay technique. In the case of these very weakly bound ligands, unambiguous detection of bound ligand can be shown by a long correlation time (23 ns, 2 degrees C) for the fluorescence anisotropy decay. Examination of the bound D-xylityl compound's fluorescence anisotropy decay at high time resolution (< 100 ps) shows rigid association, i.e. no mobility independent of the macromolecule. All bound ligands appear to be similarly positioned in the binding site. The influence of the stereoconfiguration at the 8-position found for lumazine protein parallels that previously observed for the enzyme riboflavin synthase, where the lumazines are substrates or inhibitors. This is consistent with the finding of significant sequence similarity between these proteins. The binding rigidity may have implications for the mechanism of the enzyme.
authors: Department of Biochemistry, University of Georgia, Athens 30602.
authors: J
authors: J
authors: Lee
authors: B G
authors: BG
authors: Gibson
authors: D J
authors: DJ
authors: O'Kane
authors: A
authors: A
authors: Kohnle
authors: A
authors: A
authors: Bacher
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D002241:Carbohydrates; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins; D008433:Mathematics; D008962:Models, Theoretical; D015394:Molecular Structure; D010776:Photobacterium / Q000378:metabolism; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*; D012258:Riboflavin Synthase / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D002241:Carbohydrates; D002352:Carrier Proteins; D008024:Ligands; D008164:Luminescent Proteins; D011621:Pteridines; C026648:lumazine protein, Photobacterium; C008297:lumazine; D012258:Riboflavin Synthase