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Open AccessArticle

Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore

1
Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave., St. Petersburg 194064, Russia
2
Peter the Great St. Petersburg Polytechnic University, Polytechnicheskaya str., 29, St. Petersburg 194064, Russia
*
Author to whom correspondence should be addressed.
These authors contributed to this work equally.
Int. J. Mol. Sci. 2019, 20(23), 6067; https://doi.org/10.3390/ijms20236067
Received: 7 November 2019 / Revised: 27 November 2019 / Accepted: 29 November 2019 / Published: 2 December 2019
(This article belongs to the Collection Feature Papers in Molecular Biophysics)
Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical “transparency window” of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV. View Full-Text
Keywords: bacterial phytochrome; near-infrared biomarkers; iRFP713; fluorescence quantum yield; phycocyanobilin; biliverdin bacterial phytochrome; near-infrared biomarkers; iRFP713; fluorescence quantum yield; phycocyanobilin; biliverdin
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Stepanenko, O.V.; Stepanenko, O.V.; Shpironok, O.G.; Fonin, A.V.; Kuznetsova, I.M.; Turoverov, K.K. Near-Infrared Markers based on Bacterial Phytochromes with Phycocyanobilin as a Chromophore. Int. J. Mol. Sci. 2019, 20, 6067.

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