IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Spectroscopic and functional characterization of a LOV-type photoreceptor from Xanthomonas citri subsp. citri
Autor/es:
IVANA KRAISELBURD; WOLFGANG GÄRTNER; ABA LOSI; ELENA G. ORELLANO
Lugar:
Córdoba
Reunión:
Congreso; II Reunión de Fotobiólogos Moleculares Argentinos; 2013
Resumen:
Most living organisms have the ability to sense and respond to light. Light sensing is accomplished by photoreceptors proteins carrying structures suitable for light absorption. Most biological photoreceptors have light-perception domains coupled to a variety of signaling domains which are responsible for the transduction of the light signal. LOV (Light, Oxygen, Voltage)-domain proteins are photoreceptors sensitive for the blue light region of the visible spectrum. The molecule responsible of light-sensing (chromophore) is flavin mononucleotide (FMN) which is non-covalently attached to the protein moiety, but becomes covalently bound upon the absorption of light. The covalent bound is formed by means of a conserved cysteine residue, after the light-induced conversion of the chromophore into a triplet state. The covalent form represents the active (signaling) state of the photoreceptor. This form thermally converts to the dark state of the protein. Xanthomonas citri subsp citri (Xcc) is a gram negative bacterium responsible for citrus canker, a severe disease that affects the Citrus genus. The Xcc genome sequence includes a gene encoding a LOV protein that contains an N-terminal LOV domain associated to a C-terminal histidine kinase domain and a response regulator domain (hybrid HK-RR). Previously, we confirmed that the Xcc-LOV protein is a legitimate blue light photoreceptor that is involved in the regulation of several physiological processes directly associated with the bacterial ability to colonize host plants. In this work, we studied the mechanism by which the Xcc-LOV photoreceptor is activated upon an incoming blue-light photon to start a signal transduction cascade. We generated a mutated Xcc-LOV protein, lacking the active cystein residue and unable to generate the covalent photoadduct involved in LOV photochemistry. We expressed and purified the wild type and mutant Xcc-LOV proteins and subjected them to multiple spectroscopic analyses. Steady state absorbance and fluorescent measurements where used to study the properties of the bounded chromophore and to evaluate the spectral changes in the protein upon the absorption of light. Fluorescence anisotropy studies were performed to evaluate the rigidity of the flavins environment. Changes in fluorescence emission upon photoconversion were analyzed to determine the lifetime of the excited state and the quantum yield of adduct formation. Moreover we studied the formation and decay of the intermediate triplet state in the wild type and mutant proteins by time-resolved measurements of absorption differences using flash photolysis spectroscopy and by a photocalorimetric aproach using laser-induced optoacoustic spectroscopy (LIOAS). Finally, we analyzed the functional properties of the Xcc-LOV protein. We determined the kinase activity in the dark and light states of the protein in order to evaluate the potential induction of this activity as the initial step of a cascade that results on physiological chances in the bacterium in response to light.