Structure-assisted ligand-binding analysis using fluorogenic photoaffinity labeling

Photoaffinity labeling (PAL) technique using a fluorogenic cross-linker is used to monitor the nucleotide-binding pocket within a protein. A coumarin fluorophore formed in the binding domain due to ultraviolet (UV) irradiation has been shown to accelerate the sequencing of the labeled peptide as well as identification of the labeled site by liquid chromatography (LC)-tandem mass spectrometry (MS), in addition to providing information on the ligand binding state. Selective monitoring of the predefined fluorescence peaks among the numerous digests obtained from high performance liquid chromatography (HPLC) clearly indicates the binding capability of the ligand to the entire protein as well as to the corresponding binding domain under various conditions. In the current study, ligand-binding analysis confirmed by the structural information of the binding state has been demonstrated using fluorogenic ATP/ADP photoactivatable probes under allosteric regulation of multiple substrates in the enzyme glutamate dehydrogenase (GDH).