Blue-light induced interaction of LOV domains from Chlamydomonas reinhardtii.
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.
Kutta
Roger J
RJ
Institut für Physikalische und Theoretische Chemie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
Hofinger
Edith S A
ES
Preuss
Hendrik
H
Bernhardt
Günther
G
Dick
Bernhard
B
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000378:metabolism; D056931:Cryptochromes; D003624:Darkness; D019281:Dimerization; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D011485:Protein Binding / Q000528:radiation effects; D020836:Protein Structure, Quaternary / Q000528:radiation effects; D017434:Protein Structure, Tertiary / Q000528:radiation effects; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity
D003432:Cross-Linking Reagents; D056931:Cryptochromes; D005420:Flavoproteins; D015800:Protozoan Proteins