Dynamic fluorescence study of the interaction of lumazine protein with bacterial luciferases.
The equilibrium association of lumazine protein from Photobacterium phosphoreum with luciferases from either P. phosphoreum or an aldehyde-requiring dark mutant of Vibrio harveyi is measured from changes of the rotational correlation time which is derived from the decay of the lumazine ligand's fluorescence anisotropy. The rotational correlation time of lumazine protein is 23 ns (2 degrees C, 0.25 M Pi) and is increased on addition of luciferase due to the formation of a higher molecular weight complex. The V. harveyi luciferase exhibits full competence for the association and a 1:1 stoichiometry with a Kd in the range 40-90 microM. At lower ionic strength (0.05 M Pi), the Kd increases but is reduced again by the addition of dodecanol or dimyristoyllecithin. In contrast, tetradecanal, a substrate for the bioluminescence reaction, exerts no influence on the association. The equilibration rate is found to be too slow and for both luciferases the Kd values are too high for the interaction of the native proteins to account quantitatively for the spectral shifting of the bioluminescence by lumazine protein.
Lee
J
J
Department of Biochemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
O'Kane
D J
DJ
Gibson
B G
BG
D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D008164:Luminescent Proteins; D008433:Mathematics; D008962:Models, Theoretical; D010776:Photobacterium / Q000201:enzymology / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; C026648:lumazine protein, Photobacterium; D008156:Luciferases