DEVELOPING HIGH CONCENTRATION, PH INDEPENDENT, SELECTIVE BIOSENSORS FOR REAL-TIME [GLUTAMATE] IN SYNAPTIC VESICLES

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, and impaired glutamate homeostasis appears to be a pathomechanism in brain ischemia, epilepsy or neurodegenerative diseases such as Alzheimer’s disease. However, the cellular processes that control intra- and extracellular [glutamate] have remained insufficiently understood. Glutamate is released via the exocytosis of synaptic vesicles from presynaptic nerve terminals, making [glutamate] in the presynaptic cytoplasm and in synaptic vesicles a major determinant of extracellular concentrations. Whereas great progress has been made in designing sensor for low synaptic glutamate concentrations, sensors suited for intracellular glutamate are not existing. To study glutamate concentrations inside synaptic vesicles or in the presynaptic cytoplasm of neurons sensors are required that distinguish between glutamate and aspartate, display low affinity and are insensitive to vesicular acidification. There exist multiple bacterial Glutamate Binding Protein (GluBP), and we combined computational simulations and experiments to develop fluorescent biosensors with such properties endow such proteins with the required properties. We initially identified key determinants of the binding pocket of Shigella flexneri GluBP by unbiased MD Simulation. GluBPs bind glutamate via an induced fit mechanism, so that mutations may affect glutamate binding or subsequent conformational changes. To distinguish between these two mechanisms, we combine rigorous unbiased MD simulations and Funnel Metadynamics with steady-state fluorescence measurements and kinetic analyses in stopped-flow experiments. Thus far, we have developed sensors with low glutamate affinity; we are currently aiming to improve the selectivity to glutamate and to use the sensor in in-vivo [glutamate] measurements.