Photoreversible interconversion of a phytochrome photosensory module in the crystalline state
[1];
[5];
more »
- Washington Univ., St. Louis, MO (United States)
- Rice Univ., Houston, TX (United States)
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Diamond Light Source Ltd., Oxfordshire (United Kingdom)
- SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source
- SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource
- Diamond Light Source Ltd., Oxfordshire (United Kingdom); Rutherford Appleton Lab., Didcot, Oxfordshire (United Kingdom)
A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs – Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. We conclude analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.
- Research Organization:
- SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Linac Coherent Light Source (LCLS) and Stanford Synchrotron Radiation Lightsource (SSRL); Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC)
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); National Science Foundation (NSF); Diamond Light Source; Biotechnology and Biological Sciences Research Council; Wellcome Trust; National Institute of General Medical Sciences (NIGMS); Washington University in St. Louis; Royal Society; Wellcome Investigator Award in Science; UK Government; USDOE Office of Science (SC), Biological and Environmental Research (BER); Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor; National Cancer Institute (NCI)
- Grant/Contract Number:
- AC02-05CH11231; GM127892; GM055302; GM110501; GM126289; GM117126; 1231306; 102593; AC02-76SF00515; AC02-06CH11357; 085P1000817; ACB-12002; AGM-12006; 1S10OD012289-01A1; RSWF\R2\182017; 210734/Z/18/Z
- OSTI ID:
- 1599832
- Alternate ID(s):
- OSTI ID: 1604574
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, Issue 1; ISSN 0027-8424
- Publisher:
- National Academy of SciencesCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
Similar Records
Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: Photoconversion and signal transduction
Photosensing and Thermosensing by Phytochrome B Require Both Proximal and Distal Allosteric Features within the Dimeric Photoreceptor