Synthesis of a 13C-methylene-labeled isoleucine precursor as a useful tool for studying protein side-chain interactions and dynamics

J Biomol NMR. 2024 Mar;78(1):1-8. doi: 10.1007/s10858-023-00427-2. Epub 2023 Oct 11.

Abstract

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.

Keywords: CH-π interaction; Ligand binding; Precursor labeling; Transversal relaxation studies.

MeSH terms

  • Isoleucine*
  • Ligands
  • Nuclear Magnetic Resonance, Biomolecular
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Transcription Factors* / chemistry

Substances

  • Isoleucine
  • Transcription Factors
  • Nuclear Proteins
  • Ligands