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Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis.

ABSTRACT: The carbonyl group is now a widely useful, nonproteinogenic functional group in chemical biology, yet methods for its generation in proteins have relied upon either cotranslational incorporation of unnatural amino acids bearing carbonyls or oxidative conversion (chemical or enzymatic) of existing natural amino acids. If available, alternative strategies for directly adding the C=O group through C-C bond-forming C-carbonylation, particularly at currently inaccessible amino acid sites, would provide a powerful method for adding valuable reactivity and expanding possible function in proteins. Here, following a survey of methods for HCF₂· generation, we show that reductive photoredox catalysis enables mild radical-mediated difluoromethylation-hydrolysis of native protein residues as an effective method for carbonylation. Inherent selectivity of HCF₂· allowed preferential modification of Trp residues. The resulting C-2-difluoromethylated Trp undergoes Reimer-Tiemann-type dehalogenation providing highly effective spontaneous hydrolytic collapse in proteins to carbonylated HC(O)-Trp (C-formyl-Trp = CfW) residues. This new, unnatural protein residue CfW not only was found to be effective in bioconjugation, ligation, and labeling reactions but also displayed strong "red-shifting" of its absorption and fluorescent emission maxima, allowing direct use of Trp sites as UV-visualized fluorophores in proteins and even cells. In this way, this method for the effective generation of masked formyl-radical "HC(O)·" equivalents enables first examples of C-C bond-forming carbonylation in proteins, thereby expanding the chemical reactivity and spectroscopic function that may be selectively and post-translationally "edited" into biology.

SUBMITTER: Imiolek M

PROVIDER: S-EPMC7845020 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis.

Imiołek Mateusz M Isenegger Patrick G PG Ng Wai-Lung WL Khan Aziz A Gouverneur Véronique V Davis Benjamin G BG

ACS central science 20210113 1

The carbonyl group is now a widely useful, nonproteinogenic functional group in chemical biology, yet methods for its generation in proteins have relied upon either cotranslational incorporation of unnatural amino acids bearing carbonyls or oxidative conversion (chemical or enzymatic) of existing natural amino acids. If available, alternative strategies for directly adding the C=O group through C-C bond-forming C-carbonylation, particularly at currently inaccessible amino acid sites, would provi ...[more]

PMID: 33532577

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Project description:The protein docking problem has two major aspects: sampling conformations and orientations, and scoring them for fit. To investigate the extent to which the protein docking problem may be attributed to the sampling of ligand side-chain conformations, multiple conformations of multiple residues were calculated for the uncomplexed (unbound) structures of protein ligands. These ligand conformations were docked into both the complexed (bound) and unbound conformations of the cognate receptors, and their energies were evaluated using an atomistic potential function. The following questions were considered: (1) does the ensemble of precalculated ligand conformations contain a structure similar to the bound form of the ligand? (2) Can the large number of conformations that are calculated be efficiently docked into the receptors? (3) Can near-native complexes be distinguished from non-native complexes? Results from seven test systems suggest that the precalculated ensembles do include side-chain conformations similar to those adopted in the experimental complexes. By assuming additivity among the side chains, the ensemble can be docked in less than 12 h on a desktop computer. These multiconformer dockings produce near-native complexes and also non-native complexes. When docked against the bound conformations of the receptors, the near-native complexes of the unbound ligand were always distinguishable from the non-native complexes. When docked against the unbound conformations of the receptors, the near-native dockings could usually, but not always, be distinguished from the non-native complexes. In every case, docking the unbound ligands with flexible side chains led to better energies and a better distinction between near-native and non-native fits. An extension of this algorithm allowed for docking multiple residue substitutions (mutants) in addition to multiple conformations. The rankings of the docked mutant proteins correlated with experimental binding affinities. These results suggest that sampling multiple residue conformations and residue substitutions of the unbound ligand contributes to, but does not fully provide, a solution to the protein docking problem. Conformational sampling allows a classical atomistic scoring function to be used; such a function may contribute to better selectivity between near-native and non-native complexes. Allowing for receptor flexibility may further extend these results.

Dataset Information

Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis.

Publications

Residue-Selective Protein C-Formylation via Sequential Difluoroalkylation-Hydrolysis.

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