Petra Meszaros1, Sebastian Westenhoff1,2
1Uppsala University, BMC, Department of Chemistry, Uppsala, Sweden 2University of Gothenburg, Department of Chemistry and Molecular Biology, Gothenburg, Sweden
Plants, fungi, and bacteria adjust their growth and regulate several biological processes in response to phytochrome photosensation. Therefore, accurate structural information on the phytochrome photocycle is of fundamental importance. Phytochromes are photoreceptor proteins that interconvert between two states, called Pr (red light-absorbing, at 700 nm) and Pfr (far-red light-absorbing at 750 nm). The photoconversion occurs through a number of intermediate states. Phytochromes exhibit a unique ability to respond to two different wavelengths of light. This raises the question of how a single binding pocket and a single cofactor/chromophore can support bidirectional signaling from Pr and Pfr states. The structural mechanism of photoconversion is sparsely understood. It involves the isomerization of the cofactor (biliverdin) and coinciding structural rearrangements of the surrounding amino acid residues, which then amplify into much larger structural changes. Time-resolved crystallographic studies can unravel unique structural knowledge of this mechanism at atom-scale resolution. Most phytochromes have Pr as their resting state and the only time-resolved crystallographic studies of phyotchromes have been carried out starting from the Pr state. I study the back reaction from Pfr. For this purpose, I work with the photosensory core domains of the bacteriophytochrome from Pseudomonas aeruginosa (PaBphP), which has Pfr as resting state. My aim is to study PaBphP after light activation to resolve how the intermediate states form from Pfr. To this end I have to produce well-diffracting macrocrystals first (resolution of 2A is necessary to follow the fine rearrangements around the chromophore) and then microcrystals that are suitable for time-resolved crystallography experiments.
Keywords: phytochromes; photoactivation; SFX; photoreceptors; time-resolved crystallography