| Light is an essential factor for the survival and adaptation of plants, and therefore constantly measured by multiple photoreceptors, such as the red-light (R) and far-red light (FR) sensor phytochromes. The phytochrome superfamily encompasses a group of chromoproteins that can photoconvert reversibly between two relatively stable conformers: the R-absorbing form Pr which is the ground state, and the FR-absorbing form Pfr which is biologically active. Upon exposure to R and FR, phytochromes photoconvert between Pr and Pfr, with this switch directing various light responsive events. The recent development of several 3 dimensional (3D) structures of microbial phytochromes has provided us a better understanding of their photoconversion and signaling mechanisms. In my thesis, I describe the first 3D structure of an intact phytochrome dimer by cryo-electron microscopy which reveals an extensive dimerization interface extending from the photosensory region to the C-terminal output domain. Further biochemical experiments demonstrate that dramatic conformational changes occurred along this dimerization interface during photoconversion, implying that it functions to transmit signals from the chromophore to the output module. In addition, I generated a collection of mutant bacterial phytochromes via structure-directed mutagenesis. These mutant phytochromes display variable spectral properties, such as reduced R-absorption, elimination of photoconversion, and include a group of red fluorescent proteins which could potentially work as novel fluorophores. Finally, I demonstrate the plausibility of modifying plant light-responses by structure-directed mutagenesis of plant phytochrome genes. The Arabidopsis transgenic plants expressing these engineered phytochromes show remarkably altered responsiveness to light, including reduced and increased light sensitivity. Further studies demonstrated that these engineered phytochromes have altered photochemistry and subcellular localization in Arabidopsis , which potentially result in their altered biological activity. These studies collectively should aid in the design of biofuel and food crops by altering phytochrome photoconversion and signaling. |
