EXPLORING LIGHT-SENSING AND SIGNALING MECHANISMS OF BACTERIOPHYTOCHROMES BY C

University Of Chicago, Chicago IL

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Light is a major environmental stimulus for both prokaryotes and eukaryotes. Phytochromes are red light photoreceptors that regulate a wide range of physiological processes such as seed germination, floral induction and phototaxis in plants, fungi and bacteria. Plant phytochromes (Phy) and bacteriophytochromes (Bph) use a linear tetrapyrrole (bilin) as a chromophore and photo-convert between red-absorbing (Pr) and far-red-absorbing (Pfr) states (Rockwell et al, 2006). However, molecular and mechanistic details of photoconversion and signal transduction mechanisms remain obscure. During the last proposal (#6457) period, we have successfully conducted experiments and collected data that allow us determine four different crystal structures of the photosensory domains from three distinct bacteriophytochromes (PaBphP from P. aeruginosa and RpBphP3/RpBphP2 from R. palustris) and several mutant structures in both Pr and Pfr states. We have solved the very first Pfr structure for phytochromes of any kind. We will continue to explore structural basis of signal transduction mechanisms in bacteriophytochromes using static and time-resolved crystallography as well as small-angle X-ray scattering techniques. Our specific aims during this proposal period are: 1. to determine crystal structures of full-length bacteriophytochromes (160KD) with intact sensor and effector domains (static monochromatic crystallography);2. to cryo-trap structural intermediates during Pr/Pfr photoconversion reaction pathways (static monochromatic crystallography);3. to probe early structural intermediates (in ps-ms time scale) during photoconversion at ambient temperature (time-resolved Laue crystallography);4. to explore global structural changes that transmit light signal perceived by the N-terminal sensory domains to the C-terminal effector histidine kinase domain. This requires SAXS to study samples in solution, since such global structural rearrangements are often limited in crystal lattice.