Figure 1

Strategy to obtain psd module variants. A) Site-directed mutagenesis was used to generate mutants within the Jα helix. After verification of the construct, yeast cells were transformed with the novel plasmid and subjected to cycloheximide chase analysis. B) Screening procedure to obtain mutants with altered psd module-characteristics. The plasmids pCT337 and pDS91 were used as template during random mutagenesis of the LOV2-cODC1 construct. The PCR products were combined with linearized vector (pDS90) and ligated in yeast by homologous recombination. The yeast clones were grown in darkness on selective solid medium, duplicated and either exposed to blue light or kept in darkness. The RFP fluorescence intensity of each clone was obtained for each condition. Clones with a promising dark/light ratio were selected for patch assays in comparison with the psd module (ESM356-1 + pDS90) as well as a negative control (neg c; ESM356-1 + pRS315) after growth in darkness and under blue light. At least four independent measurements were performed for each clone (lower right graph, error: s.e.m.). C) Sequence alignment of Jα helix-forming residues of LOV2 domains from different phototropins: A. thaliana phot1 (BAD94575.1 residues 569-616), Z. mays phototropin-1 (NP_001147477.1; 476-523), A. capillus phototropin (BAA95669.1|:661-708), V. faba phototropin (BAC23098.1; 540-587), P. sativum PsPK4 (AAB41023.2; 542-589), A. sativa nph1 (AAC05083.1; 507-547), and O. sativa nph1 (ABG21841.1; 202-242) were created with the software ClustalX. The secondary structure of A. thaliana LOV2 is shown on top (green arrow: β strand, orange tube: α helix) and the one of A. sativa (dark green arrow: β strand, dark orange tube: α helix) below the alignment. The numbering follows the sequence of the psd module. The grade of conservation of an amino acid is indicated using the ClustalX convention. Residues, which are mutated in psd module variants, are indicated by a red box.