Kutta, Roger J. and Hofinger, Edith S. A. and Preuss, Hendrik and Bernhardt, Günther and Dick, Bernhard (2008) Blue-light induced interaction of LOV domains from Chlamydomonas reinhardtii. Chembiochem : a European journal of chemical biology 9 (12), pp. 1931-1938.
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The phototropin from Chlamydomonas reinhardtii is a 120 kDa blue light receptor that plays a key role in gametogenesis of this green alga. It comprises two light-sensing domains termed LOV1 and LOV2 (light oxygen and voltage) and a serine/threonine kinase domain. The post-translationally incorporated chromophore is flavin mononucleotide (FMN). Upon absorption of blue light, LOV domains undergo a photocycle that activates a Ser/Thr kinase. The mechanism of this activation is still unknown. We studied the oligomerization of the recombinant LOV1 domain (amino acids 16-133) of C. reinhardtii by means of UV/Vis spectroscopy, size-exclusion chromatography (SEC), and chemical cross-linking with glutardialdehyde. The thermal back-reaction of LOV1 from the signaling state to the dark state as monitored by UV/Vis spectroscopy after an intensive blue light pulse could not be explained by a monoexponential model, although the spectra did not indicate the presence of an additional species. Therefore, we investigated the quaternary structure of the LOV1 domain by size-exclusion chromatography in the dark. This revealed an equilibrium between dimers and higher oligomers (M(W)>200 kDa) under native conditions. No monomers were detected by SEC. However, by analysis of the equilibrium by cross-linking of the protein with glutardialdehyde and subsequent SDS-PAGE, monomers and dimers were identified. Exposure of LOV1 to blue light resulted in a decrease in the monomer/dimer ratio, followed by re-equilibration in the dark. Calculation of the solvent-accessible surface area and the Conolly surfaces of the LOV1 dimers present in the crystal structure support the experimental observation that no mononomers are detected in the native state. A model is presented that accounts for a blue-light-driven change in the quaternary structure of the LOV1 domain and gives hints to the molecular basis of light activation and regulation in LOV-containing proteins.
|Institutions:|| Chemistry and Pharmacy > Institut für Physikalische und Theoretische Chemie > Chair of Chemistry III - Physical Chemistry (Light and Matter) > Prof. Dr. Bernhard Dick |
Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer)
|Projects:||GRK 640 Sensory photoreceptors in natural and artificial systems, Blaulicht-sensitive Photorezeptoren|
|Subjects:||500 Science > 570 Life sciences|
500 Science > 540 Chemistry & allied sciences
|Refereed:||Yes, this version has been refereed|
|Created at the University of Regensburg:||Yes|
|Owner:||Prof. Dr. Bernhard Dick|
|Deposited On:||12 Dec 2008 17:05|
|Last Modified:||27 Aug 2012 14:06|