(Major Center-Driven Research Project)

UC Davis: JC Lagarias, YS Su, NC Rockwell, L Shang, K Taylor, T Huser, S Fore;

Mills College: S Spiller, LR Roberts, E Castillo

Phytochromes are a family of modular photosensory proteins that regulate physiological processes in response to light. We have been studying phytochromes and related proteins to understand the photochemistry of light-activated switching and are also able to demonstrate how these unique proteins can be used as good scaffolds for fluorescent probe development. In previous years, we have focused on the cyanobacterial phytochrome Cph1 - a biliprotein that can photoconvert between two states that absorb red (~660 nm) or far-red (~710 nm) light. Using structural analysis and directed evolution methods, we discovered that a single mutation converting tyrosine-176 to histidine (YH) ablated photoconversion and conferred intense red fluorescence on Cph1, aka “phytofluor”. Strikingly, we showed that the equivalent mutation in plant phytochromes conferred dominant, light-independent activation of phytochrome signaling pathways. This dominant, constitutive YH allele could have important applications in agriculture; for example, eliminating the shade-avoidance response in plants could enable crops to be grown at higher densities. Moreover, the development of a gene-based fluorescent probe that operates in the red or near-infrared has important implications in basic research. Roger Tsien, a world expert and pioneer in the field of fluorescent proteins, recently acknowledged that phytochromes are the only photoswitchable, genetically encoded molecules to work in the far-red regime and that they will play an essential role in ultra-high resolution imaging of mammalian tissue in the future.

As we continue to improve our understanding of the photochemistry of phytochromes, we have focused on a related protein produced by Thermosynechoccus elongatus, Tlr0924 (see figure). Unlike phytochromes that are red/far-red switchable, the cyanobacteriochrome Tlr0924 uses the same chromophore but switches between states with blue and green absorbance. Tlr0924 utilizes a significantly different photochemistry from phytochromes; however, with structural analysis based on homology modeling, we have identified cysteine-499 as playing a critical role in determining the blue/green photochemi