How pH Orchestrates the Structure of the Periplasmic Glutamate-Binding Protein

L-Glutamate (L-Glu) is the major excitatory neurotransmitter in the central nervous system and plays a key role in neuronal communication, energy metabolism, and cellular development. However, excessive glutamatergic transmission can induce excitotoxicity, leading to neuronal damage and death. Beyond its physiological role, L-Glu, commonly used in the food industry as monosodium glutamate (MSG), has raised safety concerns due to its potential adverse effects, highlighting the importance of L-Glu detection in biological and food samples. In this work, we investigate the binding interactions between the glutamate-binding protein (GluB) from Corynebacterium glutamicum and L-Glu under different pH conditions using fluorescence correlation spectroscopy (FCS). GluB was labeled with CF488 and CF647 dyes, and fluorescence fluctuations were analyzed in the absence and in the presence of L-Glu. Steady-state fluorescence measurements were conducted on the unlabeled GluB, supporting FCS. They revealed pH-dependent structural changes of GluB, with conformational rearrangements at acidic pH and partial denaturation at alkaline conditions. At pH 8.0, GluB displayed a stable conformation and a measurable response to L-Glu binding. Moreover, experiments performed on the near-infrared labeled GluB-CF647 suggested a potential applicability of GluB for living cell studies.