Time ‐resolved fluorescence anisotropy with Atto 488‐labeled phytochrome Agp1 from Agrobacterium fabrum

The cartoon shows a structural model of the phytochrome Agp1 fromAgrobacterium fabrum. The colored spheres indicate positions where Cys residues were introduced for labeling with the fluorescent Atto-488. Flexibility of protein regions was determined at the different positions in the Pr and Pfr forms of the phytochrome by time-resolved fluorescence anisotropy. Green, PAS domain; blue, GAF domain; yellow, PHY domain; orange, Dhp domain of histidine kinase; magenta, ATPase domain of histidine kinase. Flexibility parameters differed significantly between the different regions and between full-length and protein without histidine kinase, but not between Pr and Pfr. AbstractPhytochromes 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 theAgrobacterium 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 anisotro...

Source: Photochemistry and Photobiology - September 7, 2023 Category: Science Authors: Afaf Elkurdi, Gernot Guigas, Latifa Hourani ‐Alsharafat, Patrick Scheerer, Gerd Ulrich Nienhaus, Norbert Krauß, Tilman Lamparter Tags: RESEARCH ARTICLE Source Type: research