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Double Covalent Bonding of Biliverdin in Near-Infrared Fluorescent Proteins Prevents Their Proteolytic Degradation

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Abstract

In this work, we analyzed how the double covalent binding of the biliverdin ligand (BV) in the near-infrared fluorescent protein iRFP670 containing two key cysteine residues affects the resistance of the biomarker to proteolytic degradation. It was previously revealed that the covalent attachment of BV to two key cysteine residues simultaneously is the reason for the highest fluorescence quantum yield of BV-containing near-infrared fluorescent proteins (NIR FPs) with two key cysteine residues compared to other BV-containing NIR FPs. Our data indicate that the covalent binding of BV in an NIR FP with two key cysteine residues simultaneously with two regions of the polypeptide chain, which, in addition, forms a figure-of-eight knot, leads to screening of many cleavage sites by the proteolytic enzymes trypsin and chymotrypsin in them. As a result, the covalent binding of BV in NIR FPs simultaneously with two key cysteine residues not only stabilizes their structure, but also increases their resistance to proteolytic degradation, which determines the cellular stability of biomarkers and is important for their use as fluorescent labels in the cell.

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Funding

The work was carried out in accordance with a state order to the Ministry of Science and Higher Education of the Russian Federation (FMFU-2021-0001).

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  1. Institute of Cytology, Russian Academy of Sciences, 194064, St. Petersburg, Russia

    Olga V. Stepanenko & Olesya V. Stepanenko

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  1. Olga V. Stepanenko
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Correspondence to Olga V. Stepanenko.

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The authors declare that they have no conflicts of interest. The authors declare that human beings or animals were not used as subjects in the experiments.

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Abbreviations: NIR–near-infrared; FPs—fluorescent proteins; BV—biliverdin; GdnHCl—guanidine hydrochloride.

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Stepanenko, O.V., Stepanenko, O.V. Double Covalent Bonding of Biliverdin in Near-Infrared Fluorescent Proteins Prevents Their Proteolytic Degradation. Cell Tiss. Biol. 17, 275–283 (2023). https://doi.org/10.1134/S1990519X23030136

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