Cell and Tissue BiologyCell and Tissue BiologyЦитология0041-37713034-6061The Russian Academy of Sciences66967010.31857/S0041377123010108GLEWOBArticlesСтатьиDouble Covalence Bonding of Biliverdin in Near-Infrared Fluorescent Protein Prevents Their Proteolitic DegradationДвойное ковалентное связывание биливердина в ближне-инфракрасных флуоресцентных белках предотвращает их протеолитическую деградациюStepanenkoOlga V.СтепаненкоОльга В.sov@incras.ruStepanenkoOlesya V.СтепаненкоОлеся В.sov@incras.ruInstitute of Cytology RASИнститут цитологии РАН0101202365110211027022025Copyright ©; 2023, Ольга В. Степаненко, Олеся В. СтепаненкоCopyright ©; 2023, Ольга В. Степаненко, Олеся В. Степаненко2023Ольга В. Степаненко, Олеся В. СтепаненкоОльга В. Степаненко, Олеся В. Степаненкоhttps://journals.eco-vector.com/0041-3771/article/view/669670

In the present work, we analyze how the double covalent binding of biliverdin ligand (BV) in the near-infrared fluorescent protein iRFP670, containing two key cysteine residues, affects the stability of this biomarker to proteolytic degradation. It has been previously found that the covalent attachment of BV simultaneously with two cysteine residues is the cause of 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 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 cysteine residues not only stabilizes their structure, but their resistance to proteolytic degradation can also increase, which determines the cellular stability of biomarkers and is important for their use as fluorescent tag in the cell.

В настоящей работе мы проанализировали как двойное ковалентное связывание лиганда биливердина (BV) в ближне-инфракрасном флуоресцентном белке iRFP670, содержащем два ключевых остатка цистеина, влияет на устойчивость биомаркера к протеолитической деградации. Ранее выявлено, что ковалентное присоединение BV одновременно к двум ключевых остаткам цистеина является причиной наибольшего квантового выхода флуоресценции BV-содержащих ближне-инфракрасных флуоресцентных белков (NIR-FP) с двумя ключевыми остатками цистеина по сравнению с остальными BV-содержащими NIR-FP. Наши данные указывают на то, что ковалентное связывание BV в NIR-FP с двумя ключевыми остатками цистеина одновременно с двумя участками полипептидной цепи, которая, помимо этого, формирует узел типа восьмерки, приводит к экранированию в них многих сайтов расщепления протеолитическими ферментами трипсином и химотрипсином. Как следствие, при ковалентном связывании BV в NIR-FP одновременно с двумя ключевыми остатками цистеина не только стабилизируется их структура, но и может возрастать их устойчивость к протеолитической деградации, что определяет клеточную стабильность биомаркеров и важно для их использования в качестве флуоресцентных меток в клетке.

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