A complex array of photoreceptors coordinates the response of organisms to their surrounding light environment. One influential set is the phytochromes (Phys), a large and diverse group of photoreversible chromoproteins that use a bilin pigment for light detection. These dimeric biliproteins sense red (R) and far-red light (FR) through two relatively stable conformational states, an R-absorbing Pr form that typically represents the ground state, and an FR-absorbing Pfr form that typically represents the activated state. By photointerconverting between Pr and Pfr, Phys act as light-regulated switches. Despite their agricultural and pathological importance and evolutionary conservation, it is not fully understood at the molecular level how Phy-type photoreceptors photoconvert between Pr and Pfr nor how this switch reports to organisms about the light around them. In the past few years, major breakthroughs have been made in understanding how Phys work at the atomic level through the determination of 3-D structural models of the photosensing module from several microbial representatives. These structures revealed the configuration of the bilin pigment and how it is cradled within its binding pocket, identified a likely route whereby plant Phys evolved from bacterial progenitors, and identified several unique structural features likely critical to signaling. During prior NSF-funded studies, the first paired ground and photoactivated state structures of the chromophore pocket from two divergent Phys were generated by nuclear magnetic resonance (NMR) spectroscopy. Comparisons of these structures provided the first glimpse into how light triggers light perception. Included are light-driven rotation of the bilin followed by rearrangement of numerous chromophore/amino acid contacts. Ultimately, these alterations must convert light energy into mechanical motion that reorganizes the output module and affect its signaling potential. This project will build upon these structural studies to answer key questions. What is the structure of a complete Phy dimer? How does rotation of the bilin followed by structural changes within the binding pocket alter Phy signaling? What is the structure of a plant Phy and how can this information be used to engineer Phy signaling for agricultural benefit? Significant to this work are the development of recombinant systems that produce large amounts of assembled photoreceptors and advances that have culminated in the generation of diffraction quality crystals of a plant Phy. Specifically, this research plan will: (1) develop more complete structures of a microbial Phy with its signal output module, (2) define how the knot, spine, and hairpin features contribute to Phy signaling, (3) determine how the distinctive cyanobacteriochrome subfamily uniquely detects other portions of the light spectrum, (4) generate an X-ray crystallographic structure of the photosensing module of a plant Phy, and (5) use a combination of NMR spectroscopic, X-ray crystallographic, and single particle electron microscopic approaches to generate models of a complete plant Phy dimer with and without signaling partners.
Broader Impacts:
This research will provide an essential framework to better understand the structure, function, and evolution of the Phy superfamily. The anticipated results will ultimately help elucidate how microorganisms and plants sense their light environment, which could have important ramifications for understanding microbial ecosystems, the control of important microbial pathogens, and for the development of new strategies to improve the productivity of food and biofuel crops. In addition, the project will enhance scientific infrastructure via a cooperative arrangement for the training of postdoctoral, graduate, undergraduate, and minority students in modern molecular and structure-based approaches in biological research. Training will also involve high school students sponsored by the Wisconsin Youth Apprenticeship Program.
