Abstract
The molecular structure of mBDFP, a far-red fluorescent protein (FPs) derived from an allophycocyanin homolog was resolved to 2.52 Å. Its biliverdin chromophore was found to be attached to the protein in an unusual way that was never observed in natural phycobiliproteins, and only once in a sub-population of artificial bacteriophytochrome-derived FPs. One of the biliverdin's vinyl groups had two cysteine residues covalently bound to its two carbon atoms. This reduces the conjugation length of the biliverdin π-electron system, which shifts the absorption and emission spectra by about 40 nm, from the near-infrared to the far-red region of the spectrum. By spectrally characterizing a set of mBDFP mutants, we show that such spectral shifts can be induced by modifying a single residue in either one of two critical positions in the vicinity of the binding cysteines. This changes the reactivity of biliverdin and the cysteine's thiols towards forming one, or two thioether bonds to the vinyl group. The ability to control the spectral properties of BDFP by specific point mutations opens many possibilities for rational design of far-red and near-infrared FPs that are of great interest to the development of fluorescence markers for bioimaging since most biological tissues are transparent in this spectral window.
Original language | American English |
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Article number | e4412 |
Journal | Protein Science |
Volume | 31 |
Issue number | 9 |
DOIs | |
State | Published - Sep 2022 |
Bibliographical note
Funding Information:The authors thank the staff at beamline P13 (DESY, EMBL, Hamburg, Germany) for kind support during screening of the crystals. The authors acknowledge the European Synchrotron Radiation Facility (Grenoble, France) for provision of synchrotron radiation facilities and the authors would like to thank the staff of ID30A-3 for their kind support during data collection. The Center for Structural Studies is funded by the Deutsche Forschungsgemeinschaft (DFG Grant number 417919780; INST 208/740-1 FUGG). The authors are grateful to Hugo Scheer, Universität München, and to Wolfgang Gärtner, Universität Leipzig, for their critical reviewing and comments. K.H.Z. (Grants 31861143029 and 31770822) is grateful for support from the National Natural Science Foundation of China. D.N. and K.H.Z. acknowledge financial support from the ISF-NSFC joint research program (Grant no. 2274/18). D.N. and I.S. acknowledge support from the Israel Ministry of Science and Technology (Grant 3-16311). I.S. has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement 678169; “PhotoMutant”).
Funding Information:
The authors thank the staff at beamline P13 (DESY, EMBL, Hamburg, Germany) for kind support during screening of the crystals. The authors acknowledge the European Synchrotron Radiation Facility (Grenoble, France) for provision of synchrotron radiation facilities and the authors would like to thank the staff of ID30A‐3 for their kind support during data collection. The Center for Structural Studies is funded by the Deutsche Forschungsgemeinschaft (DFG Grant number 417919780; INST 208/740‐1 FUGG). The authors are grateful to Hugo Scheer, Universität München, and to Wolfgang Gärtner, Universität Leipzig, for their critical reviewing and comments. K.H.Z. (Grants 31861143029 and 31770822) is grateful for support from the National Natural Science Foundation of China. D.N. and K.H.Z. acknowledge financial support from the ISF‐NSFC joint research program (Grant no. 2274/18). D.N. and I.S. acknowledge support from the Israel Ministry of Science and Technology (Grant 3‐16311). I.S. has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement 678169; “PhotoMutant”).
Publisher Copyright:
© 2022 The Protein Society.
Keywords
- allophycocyanin
- biliprotein
- biliverdin
- biomarker
- crystallization
- fluorescence