Authors:

Kalika lvaturi

Date Created:

Not specified

Course Title:

Professor:

Not specified

About Paper:

Glutamate is the primary excitatory neurotransmitter in the brain. Upon the arrival of an action potential, glutamate is released into the synaptic cleft. During this release event, the concentration of glutamate in the synaptic cleft reaches 21 mM and then rapidly returns to <20 nM within <10 ms due to uptake by neurons and glial cells. Dysregulation of glutamate levels in the synaptic cleft has been implicated in receptor-mediated excitotoxicity and is associated with numerous pathological conditions, including stroke, traumatic brain injury, glaucoma, and neurodegenerative disorders such as Alzheimer's, Huntington's, and Parkinson's diseases. The ability to accurately measure extracellular glutamate concentrations in vivo is critical for clarifying the role of glutamate in excitatory neuroplasticity and in the progression of these neurological disorders. Although numerous attempts have been made, reliable sensors capable of monitoring glutamate concentrations in vivo with high spatial resolution and in real time remain limited. To address this challenge, we aim to construct a glutamate-specific optical sensor based on a modified bacterial protein that undergoes a significant conformational change upon binding glutamate. In our design, this conformational change is translated into a change in fluorescent signal through Forster Resonance Energy Transfer (FRET). Two sites on the protein surface are selected such that the distance between them changes maximally upon glutamate binding. These positions are labeled with two different fluorophores: a FRET donor and a FRET acceptor. Binding of glutamate induces a conformational change in the protein, decreasing the distance between the donor and acceptor. This distance change modulates FRET efficiency and consequently produces a measurable change in the fluorescence intensity of the acceptor. In this research-in-progress report, we describe the development of two FRET-based sensor constructs, as well as the synthesis and purification of the fluorophore labels and the tetrazine-functionalized unnatural amino acid (UAA) fluorophore linker.

Source:

Loyola University Chicago

Topics:

No topics listed

Co-authors:

Kalika lvaturi