2 research outputs found
Phytochromes in Agrobacterium fabrum
The focus of this review is on the phytochromes Agp1 and Agp2 of Agrobacterium fabrum. These are involved in regulation of conjugation, gene transfer into plants, and other effects. Since crystal structures of both phytochromes are known, the phytochrome system of A. fabrum provides a tool for following the entire signal transduction cascade starting from light induced conformational changes to protein interaction and the triggering of DNA transfer processes
Time‐resolved fluorescence anisotropy with Atto 488‐labeled phytochrome Agp1 from Agrobacterium fabrum
Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t at any position. An intermediate t value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr