Sterically Locked Synthetic Bilin Derivatives and Phytochrome Agp1 from Agrobacterium tumefaciens Form Photoinsensitive Pr- and Pfr-like Adducts

Abstract

Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15‫؍‬C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15‫؍‬C16 double bond is in either the Z or E configuration and the C14 -C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15‫؍‬C16 Z configuration and a C14 -C15 anti conformation in the Pr form and a C15‫؍‬C16 E configuration and a C14 -C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.Phytochromes are photoreceptors of plants, bacteria, and fungi with a bilin chromophore that absorb light in the red and far-red region (1). The natural chromophore differs between species: land plants use phytochromobilin (2) and cyanobacteria use phycocyanobilin (3), whereas other bacteria use biliverdin (BV) 1 (4). The natural chromophore attaches covalently to a cysteine residue by an autocatalytical process, although in vitro adducts with non-covalently bound chromophores may also be spectrally active (5, 6). Agrobacterium phytochrome Agp1, which is used in the present study, belongs to the BV binding phytochromes and attaches its chromophore to Cys-20 (5, 7) via the ring A vinyl side chain of BV (8). Upon assembly, the red absorption maximum ( max ) of the chromophore shifts toward a longer wavelength, accompanied by an absorbance increase, to form the red-absorbing Pr. Light absorption of Pr initiates photoconversion into the Pfr form, which results in a further ϳ50 -70-nm red shift of max . Light absorption of Pfr initiates the reverse photoconversion into Pr. For plant phytochromes it has been shown that the first step of the Pr to Pfr photoconversion is a Z to E isomerization of the chromophore around the C15ϭC16 double bond (9), which occurs in the picosecond time scale (10, 11). Isomerization is ...

The Structure of Phytochrome: A Picture Is Worth a Thousand Spectra

“…The wild-type BV adduct is shown in the Pr state (solid) and as a calculated pure Pfr spectrum (dashed line). Figures were prepared from raw data kindly provided by Tilman Lamparter and Katsuhiko Inomata (Inomata et al, 2005). assembled with phycoerythrobilin, a bilin that is missing the C15,16 double bond, are intensely fluorescent, nonphotoconvertible biliproteins (Li et al, 1995;Murphy and Lagarias, 1997). In the DrBphP structure, the BV D-ring is twisted out of plane relative to the A-, B-, and C-rings.…”

Syntheses of Biliverdin Derivatives Sterically Locked at the CD‐Ring Components.

“…17 This 15Ea chromophore was also attached to the Agp1 apoprotein, and the adduct was found to be the P fr form of Agp1. 16 From these results, sterically locked chromophores will open new avenues of investigation concerning the stereochemistry and function of phytochrome chromophores both in vitro and in vivo.…”

“…Furthermore, size exclusion chromatograph of Agp1-M15 (the N-terminal 504 amino acids of Agp1) apoprotein adduct with compound 2a and the autophosphorylation of the Agp1 adduct with compound 2a showed that the stereochemistry of CD-ring moiety of P r form of natural Agp1 is 15Za. 16 Recently, we also prepared another derivative of the chromophore in which the stereochemistry of the CD-ring moiety is fixed to 15Ea. 17 This 15Ea chromophore was also attached to the Agp1 apoprotein, and the adduct was found to be the P fr form of Agp1.…”

“…The formylpyrrole derivative 11 10b was prepared starting from the commercially available 3-bromo-1-propanol (15) anhydride in the presence of catalytic amount of 4-(dimethylamino)pyridine (DMAP) in THF, followed by nitration with sodium nitrite in the presence of phloroglucinol in DMF to give 3-nitropropyl acetate (16) in 53% yield in two steps.…”

“…The solvent of the blue fraction was evaporated, and the resulting solid residue was recrystallized from CHCl 3 /hexane to afford compound 1a as a blue solid (24 mg, 80%). Decomposed above 270 C. IR (KBr) 3537,3265,3022,2944,2293,2253,1931,1443,1375,1230,1038,919,759,668 3-Nitropropyl Acetate (16). To a mixed solution of DMAP (1.952 g, 16 mmol) and 3-bromopropan-1-ol 15 (11.12 g, 80 mmol) in THF (40 mL) was added Ac 2 O (9.7 mL, 88 mmol) dropwise under N 2 at 0 C. After stirring for 30 min at 0 C and 2.5 h at room temperature, the solvent was removed under reduced pressure, and the residue was partitioned between EtOAc and water.…”