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Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
James J Chambers
Richard W Vachet
Nathan A Schnarr
Biochemistry | Molecular and Cellular Neuroscience | Organic Chemistry
Proper detection is the key to studying any processes on the cellular scale. Nowhere is this more evident than in the tight space which confines the synaptic cleft. Being able to ascertain the location of receptors on live neurons is fundamental to our understanding of not only how these receptors interact and move inside the cell but also how neurons function. Most detection methods rely on significantly altering the receptor; both tagging with a fluorescent protein or targeting the receptor by a fluorescent reporter in the form of a small molecule causes significant difficulties. These localization techniques often result in forced dimerization, unnatural movement, and at worst inactivation of the receptor.
Small molecule organic dyes provide a potential advantage because they can be structurally functionalized to target the protein of interest in a non-perturbing fashion which allows for information to be gathered about the targeted receptor. The work I initiated in the Chambers lab first focused on using a ligand directed fluorophore connected via a photo-labile linker. Through the use of epifluorescence microscopy, I determined that this probe targets glutamate receptors, however questions about subtype inclusion could not be addressed
The pharmacophore that our first probe is based on could be much more promiscuous than is presently appreciated in the field of neurobiology. Thus, we designed a new series of probes to allow for covalent modification and affinity purification of endogenous receptors. The second generation of probes set out to answer the questions left by the first. Purified proteins were subjected to SDS-PAGE analysis and could be applied to proteomic identification of receptors.
In addition to ligand directed probes, we have also initiated a project on a new, bimolecular photoaffinity probe in which the new methodology continues to develop. The initial studies were performed to ensure that our new strategy is able to be used in biological systems.
Hussey, Amanda M., "Chemical Biology-Based Probes For The Labeling Of Targets On Live Cells" (2015). Doctoral Dissertations. 429.