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Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

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Abstract

Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift. The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-“bundles” interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

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Abbreviations

CBCR:

Cyanobacteriochrome

GAF:

Acronym of cGMP phosphodiesterase, adenylyl cyclase and FhlA protein domain (SMART acc. no. SM00065)

GFP:

Green fluorescent protein

HO1:

Heme oxygenase 1

KPB:

Potassium phosphate buffer

Nostoc:

Anabaena (Nostoc) sp. PCC 7120

PCB:

Phycocyanobilin

PEB:

Phycoerythrobilin

PebS:

PEB synthase

PUB:

Phycourobilin

PVB:

Phycoviolobilin

Phy:

Phytochrome

RGS:

Red-green switchable protein encoded by rgs = slr1393.

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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, P.R. China

    Ya-Fang Sun, Jin-Guo Xu, Kun Tang, Dan Miao, Kai-Hong Zhao & Ming Zhou

  2. Max-Planck-Institute for Chemical Energy Conversion, Stiftstrasse 34–36, D-45470, Mülheim an der Ruhr, Germany

    Wolfgang Gärtner

  3. Department Biologie I, Universität München, Menzinger Str. 67, D-80638, München, Germany

    Hugo Scheer

Authors
  1. Ya-Fang Sun
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  2. Jin-Guo Xu
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  3. Kun Tang
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  4. Dan Miao
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Correspondence to Ming Zhou.

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c3pp50411e

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Sun, YF., Xu, JG., Tang, K. et al. Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin. Photochem Photobiol Sci 13, 757–763 (2014). https://doi.org/10.1039/c3pp50411e

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