Interaction of Photobacterium leiognathi and Vibrio fischeri Y1 Luciferases with Fluorescent (Antenna) Proteins:  Bioluminescence Effects of the Aliphatic Additive

Abstract

The kinetics of the bacterial bioluminescence reaction is altered in the presence of the fluorescent (antenna) proteins, lumazine protein (LumP) from Photobacterium or the yellow fluorescence proteins (YFP) having FMN or Rf bound, from Vibrio fischeri strain Y1. Depending on reaction conditions, the bioluminescence intensity and its decay rate may be either enhanced or strongly quenched in the presence of the fluorescent proteins. These effects can be simply explained on the basis of the same protein-protein complex model that accounts for the bioluminescence spectral shifts induced by these fluorescent proteins. In such a complex, where the fluorophore evidently is in proximity to the luciferase active site, it is expected that the on-off rate of certain aliphatic components of the reaction should be altered with a consequent shift in the equilibria among the luciferase intermediates, as recently elaborated in a kinetic scheme. These aliphatic components are the bioluminescence reaction substrate, tetradecanal or other long-chain aldehyde, its carboxylic acid product, or dodecanol used as a stabilizer of the luciferase peroxyflavin. No evidence can be found for the protein-protein interaction in the absence of the aliphatic component.

Protein-protein complexation in bioluminescence

“…), and K D = 0.7 µmol/L, evidence that YFP (and LumP in a less strong manner) has a catalytic effect on the luminescence reaction (Eckstein et al, 1990;Petushkov et al, 1995Petushkov et al, , 1996aPetushkov et al, , 1996b.…”

“…If included in the in vitro bacterial luciferase reaction, LumP blue-shifts the bioluminescence from its 490-nm maximum and also enhances the bioluminescence intensity (Fig. 1B) (O'Kane et al, 1991;Petushkov et al, 1996b). Lumazine protein (PDB code 3A3G) is a monomer with a spatial structure consisting of two barrels with nearly identical folds (Cα RMSD 0.90 Å, sequence identity 27%).…”

“…At micromolar concentrations of luciferase and LumP (where the average distance between unassociated molecules exceeds 1000 Å), the energy transfer has to involve the formation of a LumP-luciferase complex to bring the lumazine into proximity with the luciferase reaction site Lee, 1993). The existence of the YFP-luciferase complex is evidenced in an analogous manner (Petushkov et al, 1996a(Petushkov et al, , 1996b.…”

“…This countercharge distribution is characteristic of a variety of weak transient protein-protein complexes (Prudêncio and Ubbink, 2004;Kiel et al, 2004;Reichmann et al, 2007) and is believed to provide long-range electrostatic forces for the protein-protein interaction, which impacts on the association rate constant k A for complex formation. It is highly possible that long-range forces pre-orient LumP and luciferase molecules within an "encounter" complex, and that the successive formation of the fluorescent transient would "glue" the complex by decreasing its dissociation rate constant k D (Petushkov et al, 1996b;Schreiber et al, 2006). The fluorescent transient's breakdown would lead to dissociation of the complex allowing for the next turnover of luciferase with FMNH 2 , O 2 and aldehyde.…”

Purification and Characterization of Flavoproteins and Cytochromes from the Yellow Bioluminescence Marine Bacterium Vibrio Fischeri Strain Y1

“…We therefore resorted to a mass-action method, displacing the original flavin with excess of the lumazine derivative. Ligand exchange with certain flavin derivatives has been achieved (unpublished results) to follow up our suggestion that spectral variation would be a test of the weak coupling mechanism [4]. Bioluminescence spectral-shift experiments were not successful, however, because of the rebinding of ever-present free FMN in the in vitro bioluminescence reaction mixture.…”