| Phytochromes (Phys) are red/far-red light photoreceptors present in the bacterial, fungal and plant kingdoms where they direct many aspects of photosensory behavior, including seed germination and flowering in plants and pigment production and phototaxis in bacteria. Phys consist of an N-terminal domain which covalently binds a bilin (or tetrapyrrole) chromophore and senses light followed by a C-terminal region that typically transmits the light signal through a histidine kinase relay. To better understand how Phys detect light, my thesis research has dealt with the structural and biochemical analyses of the chromophore binding domain (CBD) of bacteriophytochromes (BphPs).; Through bilin binding assays using both mutated proteins and modified bilins, I confirmed that BphPs covalently attach their chromophore, biliverdin IXalpha (BV), to a conserved N-terminal cysteine via the A-ring vinyl side chain of BV.; By using x-ray crystallography, I solved the first three-dimensional atomic structure of a CBD, to 2.5 A resolution, using the BphP from the proteobacterium Deinococcus radiodurans (Dr). Crystallization optimization and protein engineering then enabled me to solve a second three-dimensional structure of DrCBD to 1.56 A resolution. These structures confirmed that the CBD is comprised of a PAS and GAF domain, revealed that the GAF domain cradles BV in a ZZZsyn,syn,anti conformation as Pr, and possibly identified a biologically relevant dimer interface. The most surprising aspect of the DrCBD structure is that it forms a deeply buried figure-of-eight knot where a conserved insertion within the GAF domain lassoes 34 residues near the N-terminus.; Inspection of the DrCBD models identified a possible mode of evolution within the Phy superfamily and revealed that many of the residues that directly contact the chromophore are conserved throughout this photoreceptor superfamily. Analysis of substitution mutants of Dr BphP demonstrated that bilin preference in the Phy superfamily is dependent on the position of the cysteine responsible for its covalent attachment and that many residues which directly contact the chromophore are necessary for the proper photochemical properties of DrBphP. Collectively, my thesis studies have contributed to the field of Phy research by providing the first atomic glimpse into the photochemistry and evolution of this group of photoreceptors. |
