title: A polarity probe for monitoring light-induced structural changes at the entrance of the chromophore pocket in a bacterial phytochrome.
abstract: Light-induced structural changes at the entrance of the chromophore pocket of Agp1 phytochrome were investigated by using a thiol-reactive fluorescein derivative that is covalently attached to the genuine chromophore binding site (Cys-20) and serves as a polarity probe. In the apoprotein, the absorption spectrum of bound fluorescein is red-shifted with respect to that of the free label suggesting that the probe enters the hydrophobic chromophore pocket. Assembly of this construct with the chromophores phycocyanobilin or biliverdin is associated with a blue-shift of the fluorescein absorption band indicating the displacement of the probe out of the pocket. The probe does not affect the photochromic and kinetic properties of the noncovalent bilin adducts. Upon photoconversion to Pfr, the probe spectrum undergoes again a bathochromic shift and a strong rise in CD indicating a more hydrophobic and asymmetric environment. We propose that the environmental changes of the probe reflect conformational changes at the entrance of the chromophore pocket and are indicative for rearrangements of the chromophore ring A. Flash photolysis measurements showed that the absorption changes of the probe are kinetically coupled to the formation of Meta-R(C) and Pfr. In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-RC substates. Analogous results to that of the noncovalent phycocyanobilin adduct were obtained with the mutant V249C in which probe and chromophore are covalently attached. The conformational changes of the chromophore are correlated to proton transfer to the protein surface.
authors.affiliation: Department of Physics, Biophysics Group, Freie Universität Berlin, Arnimallee 14, Berlin D-14195, Germany.
authors.forename: Berthold
authors.initials: B
authors.lastname: Borucki
authors.forename: Tilman
authors.initials: T
authors.lastname: Lamparter
mesh_terms: D016960:Agrobacterium tumefaciens / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008027:Light; D015335:Molecular Probes / Q000737:chemistry*; D010834:Phytochrome / Q000737:chemistry*; D011487:Protein Conformation / Q000528:radiation effects
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D015335:Molecular Probes; D010834:Phytochrome; D019793:Fluorescein