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

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 γCH2 to γCF2 in 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) as D-/L- forms into histone eH3.1 at site 4 (a relevant lysine to methionine oncomutation site), and each can be detected by 19F-nuclear magnetic resonance of the γCF2 group, 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 γCF2 as 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 by 19F-based detection methods.

Pathogen-sugar interactions revealed by universal saturation transfer analysis.

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.