Abstract
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. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.
Highlights
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
We postulated that CBCRs could be ideal sources because their isolated GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domains are often sufficient for full photoconversion [1, 2]
Guided by previous crystallographic models of the TePixJ GAF domain [6], we engineered a minimal construction of the photosensory module for recombinant expression (Fig. 1 A and B)
Summary
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. 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 Analysis of these crystals by SFX should enable structural characterization of the early events that drive phytochrome photoconversion. Novel Phys have evolved to detect light–color pairs other than red/far red, including ultraviolet/blue, blue/green, or blue/orange, or to prefer Regardless of their absorption ranges, photoexcitation of Phys is thought to trigger a reversible 15Za⇋15Ea isomerization of the bilin. This rotation alters its extensive π-bond conjugation system and its light-absorption properties, with the ratio of dark-adapted and photoactivated states governed by the spectral quality of the light environment [1, 2]. While various spectroscopic studies have identified several spectrally distinct intermediates that arise on pico- to millisecond timescales (e.g., refs. 12 and 14–17) and X-ray scattering studies have observed lifetimes of small- and large-scale structural changes along various photoconversion pathways (e.g., refs. 18 and 19), the precise identity and sequence of the structural changes that follow photoexcitation remain unresolved due to a lack of high-resolution structural information from intermediates
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