biostudies-literatureUnknown78(1)Hofurthner TUniversity of ViennaChristian Doppler ForschungsgesellschaftIn this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum ¹³C-¹ H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased ¹³C-T₂ times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.Journal of biomolecular NMR1-8https://www.ebi.ac.uk/biostudies/studies/S-EPMC10981609biostudies-literatureSynthesis of a ¹³C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics.PMC10981609Mayer MToscano GLichtenecker RKontaxis GWeinstabl HHofurthner TKonrat RGeist LMcConnell DBBeier AfalseSynthesis of a ¹³C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics.In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum ¹³C-¹ H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased ¹³C-T₂ times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes.2024-01-01T00:00:00Z2024 Mar2026-07-16T12:14:48.899Z2025-04-04T19:58:07.196ZS-EPMC109816093781693310.1007/s10858-023-00427-2