Yellow light emission of Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow fluorescent protein.

Abstract

A strain of luminous bacteria, Vibriofischer Y-1, emits yellow light rather than the blue-green emission typical of other luminous bacteria. The yellow emission has been postulated previously to result from energy transfer from an electronically excited species formed in the bacterial luciferase-catalyzed reaction to a secondary emitter protein, termed the yellow fluorescent protein (YFP). We report here the purification of YFP to homogeneity without loss of the chromophore. The protein was found to be a homodimer of Mr 22,000 subunits with one weakly bound FMN per subunit. The FMN-protein complex was stabilized by 10% (vol/vol) glycerol in the buffers, allowing purification ofthe active holo-YFP. The protein migrated as a single spot with an isoelectric point of =6.5 on two-dimensional polyacrylamide gel electrophoresis and gave an N-terminal sequence of Met-Phe-Lys-Gly-Ile-ValGlu-Gly-Ile-Gly-Ile-Ile-Glu-Lys-Ile. Addition of purified YFP to a reaction in which luciferase was supplied with FMNH2 (reduced FMN) by a NADH:FMN oxidoreductase resulted in a dramatic enhancement in the intensity of bioluminescence and an additional peak in the emission spectrum at about 534 nm. The resulting bimodal bioluminescence emission spectrum had peaks at 484 nm, apparently due to emission from the luciferase-flavin complex, and at 534 nm, corresponding to the fluorescence emission maximum of YFP. This bimodal spectrum closely matched the emission spectrum in vivo. (17). This paper reports the purification to homogeneity ofthe yellow fluorescent protein, determination of its physical properties, and the optimum conditions for yellow light emission.

Protein-protein complexation in bioluminescence

“…The emission spectra of some bioluminescent bacteria are found blue or red shifted from that of the purified luciferase in vitro reaction, due to accompanying antenna proteins ( Fig. 1B and 1C) (Daubner et al, 1987;Lee, 1993). These proteins, lumazine protein and yellow-fluorescent protein (YFP), contain a highly fluorescent ligand (6,7-dimethyl-8-ribityllumazine, FMN, or riboflavin), and apparently associate with a high-energy intermediate in the luciferase reaction so that the excitation step involves a Förster-type coupling to the antenna transition, and the emission becomes that of the bound lumazine or flavin (Petushkov and Lee, 1997).…”

“…Shifted in vitro spectra closely resemble the in vivo bioluminescence of the bacterial species containing the corresponding antenna proteins (based on Shimomura (2006)). (C) Cultures of P. phosphoreum (left) and V. fischeri Y-1 (right) (from Daubner et al (1987) reprinted with permission of use from the publisher). (D) Reaction mechanism in bacterial bioluminescence.…”

“…YFP contains riboflavin or flavin as a fluorophore and modulates the blue bioluminescence color of the in vitro Y-1 luciferase reaction to the yellow (maximum 542 nm) (Daubner et al, 1987;Macheroux et al, 1987;Baldwin et al, 1990;Petushkov and Lee, 1997). LumP and YFP are homologous (37%) (O'Kane et al, 1991) and also share homology with riboflavin synthase, which binds two molecules of the lumazine (O' Kane and Prasher, 1992).…”

“…Another accessory protein, NADPH:FMN oxidoreductase, supplies FMNH 2 for reaction with oxygen and aldehyde in the active site of bacterial luciferase (Jeffers et al, 2003). The function of bioluminescence color modulation belongs to the so-called antenna proteins, such as green-fluorescent proteins (GFP) in the bioluminescent coelenterates (Morin and Hastings, 1971;Ward and Cormier, 1979), or lumazine protein and yellow-fluorescent protein in bioluminescent bacteria Daubner et al, 1987;Macheroux et al, 1987). Fluorescence properties of antenna proteins are due to a bound fluorophore, a molecule known to be involved in intermediary metabolism (bacterial antenna proteins), or a moiety autocatalytically synthesized from amino acid residues of the apo-protein (GFP).…”

Time‐Resolved Fluorescence Study of the Dissociation of FMN from the Yellow Fluorescence Protein from Vibrio fischeri

“…This strongly emitting protein tumed out to be a flavoprotein with flavin mononucleotide (FMN) (3,4). In addition, a closely related protein containing riboflavin as the chromophore has been identified (5,6).…”