Fluorescence of Phytochrome Adducts with Synthetic Locked Chromophores

Abstract

We performed steady state fluorescence measurements with phytochromes Agp1 and Agp2 of Agrobacterium tumefaciens and three mutants in which photoconversion is inhibited. These proteins were assembled with the natural chromophore biliverdin (BV), with phycoerythrobilin (PEB), which lacks a double bond in the ring C-D-connecting methine bridge, and with synthetic bilin derivatives in which the ring C-D-connecting methine bridge is locked. All PEB and locked chromophore adducts are photoinactive. According to fluorescence quantum yields, the adducts may be divided into four different groups: wild type BV adducts exhibiting a weak fluorescence, mutant BV adducts with about 10-fold enhanced fluorescence, adducts with locked chromophores in which the fluorescence quantum yields are around 0.02, and PEB adducts with a high quantum yield of around 0.5. Thus, the strong fluorescence of the PEB adducts is not reached by the locked chromophore adducts, although the photoconversion energy dissipation pathway is blocked. We therefore suggest that ring D of the bilin chromophore, which contributes to the extended -electron system of the locked chromophores, provides an energy dissipation pathway that is independent on photoconversion.

Structure-guided Engineering Enhances a Phytochrome-based Infrared Fluorescent Protein

“…Several endeavors into this realm have been made. Incorporation of the non-native tetrapyrrole phycoerythrobilin into plant or bacterial phytochromes can lead to intensely fluorescent proteins (26,27). A Y176H change in Cph1 renders it fluorescent (28,29), and the equivalent substitution in Arabidopsis thaliana phytochromes A and B confers fluorescence and a constitutive red light phenotype (28,30).…”

Red-light absorption and fluorescence of phytochrome chromophores: A comparative theoretical study

“…In the experimental reference study [25], five different locked bilin chromophores with either one (between rings C and D) or two (between both rings C and D and rings A and B) cyclic locks were considered. Furthermore, the absorption and fluorescence measurements were done at both pH = 1 and pH = 7.8 [25].…”

“…Specifically, we present a systematic investigation of how well different density functional theory (DFT) and ab initio quantum chemical methods reproduce the unique set of experimental absorption and emission maxima of free bilin chromophores in solution recently recorded by Zienicke et al [25]. These authors were able to overcome the difficulty in measuring the weak fluorescence of bilins [26] by synthesizing sterically locked chromophores in which the competing photoisomerization of the C15=C16 bond is hindered (see Fig.…”

“…In this work, we take advantage of current advances in synthetic bilin chemistry [25] and present a theoretical study that provides valuable insight into the applicability of computational methods in the development of improved fluorescent proteins based on phytochromes. Specifically, we present a systematic investigation of how well different density functional theory (DFT) and ab initio quantum chemical methods reproduce the unique set of experimental absorption and emission maxima of free bilin chromophores in solution recently recorded by Zienicke et al [25].…”

Assembly of Synthetic Locked Phycocyanobilin Derivatives with Phytochrome in Vitro and in Vivo in Ceratodon purpureus and Arabidopsis

“…Recently, several synthetic locked chromophores have been assembled with bacterial phytochromes Agp1 and Agp2 in vitro to study the stereochemistry of their chromophores during the photocycle (Inomata et al, 2005;Inomata et al, 2006;Seibeck et al, 2007;Inomata et al, 2009;Zienicke et al, 2011). Adducts with 15ZaBV, in which the ring C-D-connecting methine bridge is fixed in a Z anti (Za) stereochemistry, are spectrally similar to the Pr form, whereas the 15EaBV adducts with an E anti (Ea) -configured methine bridge are comparable with the Pfr form.…”