Abstract
- ASU
- asymmetric unit
- BV, PCB, PVB (bilin compounds serving as chromophores)
- biliverdin Ixα, phycocyanobilin, phycoviolobilin
- CAPSO
- N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid
- CBCR
- cyanobacteriochrome
- GAF (protein domain)
- cGMP-specific phosphodiesterases adenylyl cyclases and FhlA
- IMAC
- immobilized metal-affinity chromatography
- MR
- molecular replacement
- PAS (protein domain)
- Per-Arnt-Sim
- PHY (protein domain)
- phytochrome-specific
- Pfr, Pg, Pr
- far red-, green-, and red-absorbing states of phytochromes and CBCRs
Summary The GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) with an in vivo assembled phycocyanobilin (PCB) chromophore has been crystallized in parental state (1.8 Å) and photoproduct state (1.86 Å), identified by 15-Z and 15-E chromophore configuration. Comparison of both structures for the same protein allows precise determination of structural changes after photo-activation. The chromophore photoisomerization causes an outward movement and partial helix formation of a formerly unstructured loop. A tryptophan residue located in this loop, in π-π stacking distance to PCB in the dark state, moves away by 14 Å opening the binding cleft for the entry of water molecules. Also the in vitro assembled protein (chromophore addition to apo-protein) has been crystallized (1.6 Å resolution). Most importantly, an intermediate structure was solved (2.1 Å) with the protein in photoproduct conformation and the chromophore already isomerized into the parental 15-Z configuration, thereby giving insight into chromophore-initiated conformational protein changes.
Impact Statement This manuscript presents crystal structures of a photochromic protein in both states, before (1.6 Å) and after (1.9 Å) the light induced photochemical event with sufficient resolution to allow detailed description of conformational changes of chromophore and protein. The light driven reaction, double bond photoisomerization of a covalently bound bilin chromophore is presented here for the first time. Our results allow determining the impact of the chromophore photochemistry on the protein conformation. In addition, we succeeded in trapping an intermediate carrying the chromophore already in isomerized state with the protein still in unchanged conformation. Absorption spectra of this intermediate clearly demonstrate a color change, thus allowing conclusion that the absorption of phytochromes is predominantly determined by the chromophore conformation alone with only moderate effect of the surrounding protein.
Authors’ Contributions XX, KHZ, and WG designed the experiment. XX generated the protein. AH performed crystallization trials, collected the X-ray diffraction data and solved the structure. All authors contributed in preparing the manuscript.
