Posttranslational, site-directed photochemical fluorine editing of protein sidechains to probe residue oxidation state via 19F-nuclear magnetic resonance

Autor: Isenegger, PG, Josephson, B, Gaunt, B, Davy, MJ, Gouverneur, V, Baldwin, AJ, Davis, BG
Rok vydání: 2023
Předmět:
Zdroj: Nature Protocols. 18:1543-1562
ISSN: 1750-2799
1754-2189
DOI: 10.1038/s41596-022-00800-9
Popis: The fluorination of amino acid residues represents a near-isosteric alteration with the potential to report on biological pathways, yet the site-directed editing of carbon–hydrogen (C–H) bonds in complex biomolecules to carbon–fluorine (C–F) bonds is challenging, resulting in its limited exploitation. Here, we describe a protocol for the posttranslational and site-directed alteration of native γCH2to γCF2in protein sidechains. This alteration allows the installation of difluorinated sidechain analogs of proteinogenic amino acids, in both native and modified states. This chemical editing is robust, mild, fast and highly efficient, exploiting photochemical- and radical-mediated C–C bonds grafted onto easy-to-access cysteine-derived dehydroalanine-containing proteins as starting materials. The heteroaryl–sulfonyl reagent required for generating the key carbon-centered C• radicals that install the sidechain can be synthesized in two to six steps from commercially available precursors. This workflow allows the nonexpert to create fluorinated proteins within 24 h, starting from a corresponding purified cysteine-containing protein precursor, without the need for bespoke biological systems. As an example, we readily introduce three γCF2-containing methionines in all three progressive oxidation states (sulfide, sulfoxide and sulfone) asD-/L- forms into histone eH3.1 at site 4 (a relevant lysine to methionine oncomutation site), and each can be detected by19F-nuclear magnetic resonance of the γCF2group, as well as the two diastereomers of the sulfoxide, even when found in a complex protein mixture of all three. The site-directed editing of C–H→C–F enables the use of γCF2as a highly sensitive, ‘zero-size-zero-background’ label in protein sidechains, which may be used to probe biological phenomena, protein structures and/or protein–ligand interactions by19F-based detection methods.
Databáze: OpenAIRE
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