Assembly of Synthetic Locked Chromophores with Agrobacterium Phytochromes Agp1 and Agp2

Abstract

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15‫؍‬C16 double bond is fixed in either the E or Z configuration and the C14 -C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4‫؍‬C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4‫؍‬C5 Z C5-C6 syn C15‫؍‬C16 Z C14 -C15 anti stereochemistry and that in the Pfr chromophore the C15‫؍‬C16 double bond has isomerized to the E configuration, whereas the C14 -C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.Almost all organisms have adopted mechanisms to sense light via photoreceptors and respond to different light conditions in manifold ways. Phytochromes are photoreceptors that are reversibly converted by light between two stable or long lived forms, the red-absorbing Pr 3 and the far-red-absorbing Pfr (1). Depending on the species, phytochromes bind one of three bilins as a chromophore: land plants utilize phytochromobilin (P⌽B) (2), cyanobacteria use phycocyanobilin (PCB) (3, 4), whereas all other organisms including proteobacteria and fungi use biliverdin (BV) (5-7). The chromophore is covalently attached to a cysteine residue during an autocatalytic process. Whereas plant and cyanobacterial phytochromes use a cysteine in the so-called GAF domain of the protein as chromophore attachment site (2), the chromophore-binding cysteine of BVbinding phytochromes is close to the N terminus of the protein (8 -10). NMR studies with plant phytochromes showed that the chromophore undergoes isomerization around the double bond connecting the rings C and D during photoconversion from Pr to Pfr (11). The configuration of this double bond is Z in the Pr and E in the Pfr form. Time-resolved spectroscopy techniques show that this Z-E isomerization, leading to the lumi-R photoproduct, takes place on a picosecond time scale (12, 13). Later intermediates, termed meta-Ra, meta-Rb, and meta-Rc, as well as the photoproduct Pfr are formed in the microsecond and millis...

High Resolution Structure of Deinococcus Bacteriophytochrome Yields New Insights into Phytochrome Architecture and Evolution

“…see Ref. 52) to define the movements of the chromophore and protein during the initial fast photochemical events predicted to follow R absorption by Pr (3,34,53). Our surface engineering strategy to generate well ordered crystals by minimizing conflicts between symmetry mates also illustrates the utility of this approach to improve x-ray diffraction resolution.…”

Studies on the Structure and Function of Phytochromes as Photoreceptors Based on Synthetic Organic Chemistry

“…To see whether and how locked chromophores are incorporated into Agp2, the set of the above mentioned chromophores was also tested with purified recombinant Agp2. 160 Interesting differences between Agp1 and Agp2 and between different chromophores were found for the assembly rates. The slowest assembly with Agp1 was found for the 15Ea chromophore.…”

“…Results with the 5Zs and 5Za chromophores are summarized as follows: 160 (1) Both chromophores form covalent bonds with either Agp1 or Agp2. (2) The spectra and the absorption maxima of the 5Zs adducts are similar to those of the Pr form of the 18EtBV control.…”

“…20), were recently synthesized in a similar manner described for the chromophores locked at the C15 position. They were also used for assembly with Agp1 and Agp2 160 and provided deeper insight into the similarities and differences between a more conventional phytochrome Agp1 and a phytochrome Agp2, which converts to Pfr in the dark, giving further clues for the understanding of chromophore stereochemistry in the ground state and during photoconversion.…”

“…The assembly rates for 15Ea/15Za for both phytochromes are in line with the finding that Agp1 adopts a Pr form and Agp2 adopts a Pfr form in the dark. 160 For analyses on the stereochemistry of the C5-C6 single bond, new locked chromophores 5Zs and 5Za, in which C4=C5 double bond is fixed in the Z configuration and the C5-C6 single bond is fixed in either the syn or anti conformation (Fig. 20), were recently synthesized in a similar manner described for the chromophores locked at the C15 position.…”