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¹⁹F multiple-quantum coherence NMR spectroscopy for probing protein-ligand interactions.

ABSTRACT: A new ¹⁹F NMR method is presented which can be used to detect weak protein binding of small molecules with up to mM affinity. The method capitalizes on the synthetic availability of unique SF₅ containing compounds and the generation of five-quantum coherences (5QC). Given the high sensitivity of 5QC relaxation to exchange events (i.e. reversible protein binding) fragments which bind to the target with weak affinity can be identified. The utility of the method in early stage drug discovery programs is demonstrated with applications to two model proteins, the neurotoxic NGAL and the prominent tumor target β-catenin.

SUBMITTER: Zawadzka-Kazimierczuk A

PROVIDER: S-EPMC9091488 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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19F multiple-quantum coherence NMR spectroscopy for probing protein-ligand interactions.

Zawadzka-Kazimierczuk Anna A Somlyay Mate M Kaehlig Hanspeter H Iakobson George G Beier Petr P Konrat Robert R

RSC advances 20181205 71

A new 19F NMR method is presented which can be used to detect weak protein binding of small molecules with up to mM affinity. The method capitalizes on the synthetic availability of unique SF5 containing compounds and the generation of five-quantum coherences (5QC). Given the high sensitivity of 5QC relaxation to exchange events (i.e. reversible protein binding) fragments which bind to the target with weak affinity can be identified. The utility of the method in earl ...[more]

PMID: 35557931

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Project description:Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.

Dataset Information

¹⁹F multiple-quantum coherence NMR spectroscopy for probing protein-ligand interactions.

Publications

<sup>19</sup>F multiple-quantum coherence NMR spectroscopy for probing protein-ligand interactions.

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