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Near infrared fluorescent biliproteins generated from bacteriophytochrome AphB of Nostoc sp. PCC 7120

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Abstract

The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1–321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore.

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Abbreviations

AphB:

Bacterial phytochrome of Nostoc sp. PCC 7120

BV:

Biliverdin

Bph:

Bacteriophytochrome

GAF:

Protein domain (SMART acc. no. SM00065; acronym of common protein domain from cGMP phosphodiesterase, adenylyl cyclase and FhlA)

HO:

Heme oxygenase

KPB:

Potassium phosphate buffer

NIR:

Near infrared

PAS:

Protein domain (SMART acc. no. SM00091; acronym of common protein domain from period circadian protein, Ah receptor nuclear translocator protein and single-minded protein domain)

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Authors and Affiliations

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

    Che Yuan, Hui-Zhen Li, Kun Tang, Ming Zhou & Kai-Hong Zhao

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

    Kun Tang & Wolfgang Gärtner

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

    Hugo Scheer

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  1. Che Yuan
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  2. Hui-Zhen Li
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  3. Kun Tang
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  4. Wolfgang Gärtner
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  5. Hugo Scheer
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  6. Ming Zhou
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  7. Kai-Hong Zhao
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Correspondence to Ming Zhou or Kai-Hong Zhao.

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c5pp00442j

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Yuan, C., Li, HZ., Tang, K. et al. Near infrared fluorescent biliproteins generated from bacteriophytochrome AphB of Nostoc sp. PCC 7120. Photochem Photobiol Sci 15, 546–553 (2016). https://doi.org/10.1039/c5pp00442j

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