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<p>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 <i>Corynebacterium glutamicum</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月9日

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  • Giovanni Ferrara,
  • Antonio Varriale,
  • Sabato D’Auria,
  • Maria Staiano

摘要

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.