In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore

Abstract

Phytochrome B (PhyB), one of the major photosensory chromoproteins in plants, mediates a variety of light-responsive developmental processes in a photoreversible manner. To analyze the structural requirements of the chromophore for the spectral properties of PhyB, we have designed and chemically synthesized 20 analogs of the linear tetrapyrrole (bilin) chromophore and reconstituted them with PhyB apoprotein (PHYB). The A-ring acts mainly as the anchor for ligation to PHYB, because the modification of the side chains at the C2 and C3 positions did not significantly influence the formation or difference spectra of adducts. In contrast, the side chains of the B-and C-rings are crucial to position the chromophore properly in the chromophore pocket of PHYB and for photoreversible spectral changes. The side-chain structure of the D-ring is required for the photoreversible spectral change of the adducts. When methyl and ethyl groups at the C17 and C18 positions are replaced with an n-propyl, n-pentyl, or n-octyl group, respectively, the photoreversible spectral change of the adducts depends on the length of the side chains. From these studies, we conclude that each pyrrole ring of the linear tetrapyrrole chromophore plays a different role in chromophore assembly and the photochromic properties of PhyB.

Structural requirement of bilin chromophore for the photosensory specificity of phytochromes A and B

“…Although it is tempting to speculate on the existence of PCB in higher plants, this has not been identified. Interestingly, the binding efficiency of various bilins to recombinant PHYB was determined in vitro, with the result that PCB bound to PHYB more efficiently than P⌽B (21). In the present work, we found that PCB and P⌽B restore PhyB-mediated physiological responses.…”

“…R͞FR and FR͞R were generated by using a custom-built LED irradiation system, as previously reported (11 Bilin Preparation. PCB (18), P⌽B (17), and their analogs (21,29) were chemically synthesized, as reported. The bilins were stored as 2 mM stock solutions in dimethyl sulfoxide under nitrogen atmosphere at Ϫ80°C.…”

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

“…10), including natural PÈB and PCB, and reconstituted them with recombinant Arabidopsis PhyB apoprotein (PHYB) through collaborative study with biologists. 125 The difference absorption spectra of the in vitro-assembled adducts of the synthetic bilin chromophores with PHYB were measured on actinic far-red and red light irradiations. The adduct with PCB showed a similar difference spectrum to that of the PÈB adduct, although its peaks were slightly blue shifted due to the difference of the length of -conjugation system when compared with the PÈB adduct corresponding to the natural phytochrome, [137][138][139] as shown in Fig.…”

“…As mentioned above, we established an efficient and flexible synthetic method of various kinds of bilin chromophores, [46][47][48][49][50][51] including PÈB, 49 PCB, 47,48 and more than 20 kinds of PCB derivatives, 50,51 in free acid forms, which made it possible to assemble them with the apoproteins not only in vitro to analyze the spectral properties of the resulting holoproteins, 125 but also in vivo to observe their physiological functions 126 as will be seen later.…”

“…Binding efficiency of each analog by using zinc blot analysis was reported in our original report. 125 The A-ring acts mainly as the anchor for ligation to PHYB, because the modification of the side chains at the C2 and C3 positions did not significantly influence the formation (based on the results from zinc blot analysis) or difference spectra of adducts (Figs. 12a and 12b) The difference spectra were obtained by subtracting the absorption spectra recorded after red light irradiation from that recorded after far-red light irradiation.…”