Properties of the bimodal fluorescent protein produced by Photobacterium phosphoreum.
A fluorescent protein isolated from the deep-sea luminous bacterium Photobacterium phosphoreum strain bmFP has been purified, cloned and sequenced. The protein is 96.5% identical in amino acid sequence to FP390, the weakly fluorescent flavoprotein encoded by the luxF gene characteristic of Photobacterium species. Similar to FP390, bmFP is a dimer of two homologous subunits binding four FMN-myristate chromophores but has the distinctive feature of emitting a bimodal fluorescence with maxima at about 488 and 517 nm, hence the name bmFP. For both bands of this fluorescence, the excitation spectrum exhibits a peak at 336 nm, not corresponding to its flavin-like absorption spectrum. Heating of bmFP in urea resulted in a decrease in the intensity of the 488 nm band along with the appearance of a new fluorescence peaking at 423 nm, partially reversible upon the removal of the urea. Upon complete denaturation, either by heat or guanidium chloride at 65 degrees C, fluorescence characteristic of both free flavin and this 423 nm species appears. It is speculated that chromophores in different states of protonation, associated with a single protein, are responsible for the unusual spectral properties of bmFP.
Karatani
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H
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Japan. karatani@ipc.kit.ac.jp
Konaka
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T
Katsukawa
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C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D005420:Flavoproteins / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010776:Photobacterium / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D005420:Flavoproteins; D008164:Luminescent Proteins; C404984:bimodal fluorescent protein, Photobacterium phosphoreum; C081175:non-fluorescent flavoprotein, Photobacterium