Shining a light on substrate affinity of glutamate-binding protein. Single-molecule insights into pH-modulated glutamate binding.

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 investigated the binding interactions between the glutamate-binding protein (GluB) from Corynebacterium glutamicum and L-Glu under different pH conditions using fluorescence correlation spectroscopy (FCS) and steady-state fluorescence measurements. GluB was labeled with CF488 and CF647 dyes, and the fluorescence fluctuations were analyzed in the absence and in the presence of L-Glu. Steady-state fluorescence measurements were conducted on unlabeled GluB. The obtained results revealed the presence of pH-dependent structural changes of GluB at acidic pH as well as a partial denaturation of GluB structure at alkaline conditions. At pH 8.0, GluB displayed a stable structure and a measurable response to L-Glu binding. Moreover, experiments performed on GluB labeled with a near-infrared dye (GluB-CF647) suggested the potential applicability of GluB for investigations of cellular and environmental interests.