title: Fluorescence properties of the chromophore-binding domain of bacteriophytochrome from Deinococcus radiodurans.
abstract: Fluorescent proteins are versatile tools for molecular imaging. In this study, we report a detailed analysis of the absorption and fluorescence properties of the chromophore-binding domain from Deinococcus radiodurans and its D207H mutant. Using single photon counting and transient absorption techniques, the average excited state lifetime of both studied systems was about 370 ps. The D207H mutation slightly changed the excited state decay profile but did not have a considerable effect on the average decay time of the system or the shape of the absorption and emission spectra of the biliverdin chromophore. We confirmed that the fluorescence properties of both samples are very similar in vivo and in vitro. However, we found that the paraformaldehyde fixation of the Escherichia coli cells containing the recombinant phytochrome protein significantly changed the fluorescence properties of the chromophore-binding domain. The biliverdin fluorescence was diminished almost completely, and the fluorescence originated only from the protoporphyrin molecules. Our results emphasize that the effect of protoporphyrin IXa should not be ignored in the fluorescence experiments with phytochrome systems while designing better red fluorescence markers for cellular imaging.
authors: Nanoscience Center, Department of Biological and Environmental Science, University of Jyväskylä , P.O. Box 35, 40014 Jyväskylä, Finland.
authors: Heli
authors: H
authors: Lehtivuori
authors: Ilona
authors: I
authors: Rissanen
authors: Heikki
authors: H
authors: Takala
authors: Jaana
authors: J
authors: Bamford
authors: Nikolai V
authors: NV
authors: Tkachenko
authors: Janne A
authors: JA
authors: Ihalainen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001664:Biliverdine / Q000737:chemistry; D034301:Deinococcus / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001664:Biliverdine