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Photochemical Tools for Fluorescent Labeling of Endogenous Proteins

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dc.contributor.advisorJames Chambers
dc.contributor.advisorVincent Rotello
dc.contributor.advisorNathan Schnarr
dc.contributor.advisorAlejandro Heuck
dc.contributor.authorMcCarron, Stephen T
dc.contributor.departmentUniversity of Massachusetts Amherst
dc.date2024-03-27T19:09:48.000
dc.date.accessioned2024-04-26T16:09:10Z
dc.date.available2024-04-26T16:09:10Z
dc.date.submittedSeptember
dc.date.submitted2015
dc.description.abstractThe study of the dynamic movements of membrane bound proteins is typically achieved through an exogenously applied fluorescent tag or genetic modification of a receptor of interest to spatiotemporally monitor protein location. Techniques often used for labeling proteins include overexpression of a fluorescent protein such as GFP fused to a target protein or the application of antibodies. These methods benefit from superb specificity towards a receptor of interest, but may impose unforeseen consequences when studying natural protein movements. Thus, it is advantageous to development small, modular probes that would allow for visualization of endogenous membrane bound receptors in a minimally perturbed state. Several strategies employing photochemical small molecules were pursued to label important neuronal receptor targets on live cells. One method, photoaffinity labeling, requires a highly specific ligand outfitted with a UV photoreactive group and an orthogonal handle to covalently bind the ligand to the receptor for experimental analysis of the target. Using this technique we synthesized high affinity photoaffinity labels to study sigma receptors, which are believed to be involved in several neurodegenerative disorders and cancer pathologies. Another method is fluorescently tethered small molecule ligands that utilize a pharmacophore covalently bound to a fluorophore for specificity and subsequent visualization of a receptor of interest. We have designed small, minimally-perturbing modular probes that enable labeling and subcellular visualization of endogenous receptors on live cells. This technology allows for silent labeling of a receptor of interest, which allows for the receptor to be tagged with a fluorescent moiety in a non-ligated and thus active state. Using these probes we labeled the presynaptic dopamine and serotonin transporters and NMDA receptors on live cells. Finally, we have developed a method for facile alkylation of biological substrates utilizing a small, photoactivatable, electrophilc moiety that is compatible with aqueous buffer. We demonstrated that the molecule is reactive only in the presence of UV light, which gives the user temporal control over the alkylation of a nucleophile. In sum, these small molecule probes will provide chemical biologists novel tools for labeling membrane-bound receptors to visualize their location and trafficking, which will give fundamental insight into their endogenous activity.
dc.description.degreeDoctor of Philosophy (PhD)
dc.description.departmentChemistry
dc.identifier.doihttps://doi.org/10.7275/7379842.0
dc.identifier.orcidN/A
dc.identifier.urihttps://hdl.handle.net/20.500.14394/19674
dc.relation.urlhttps://scholarworks.umass.edu/cgi/viewcontent.cgi?article=1470&context=dissertations_2&unstamped=1
dc.source.statuspublished
dc.subjectPhotochemistry
dc.subjectprotein labeling
dc.subjectfluorescence
dc.subjectfluorescent probes
dc.subjectOrganic Chemistry
dc.titlePhotochemical Tools for Fluorescent Labeling of Endogenous Proteins
dc.typeopenaccess
dc.typearticle
dc.typedissertation
digcom.contributor.authorisAuthorOfPublication|email:smccarron87@gmail.com|institution:University of Massachusetts Amherst|McCarron, Stephen T
digcom.identifierdissertations_2/459
digcom.identifier.contextkey7379842
digcom.identifier.submissionpathdissertations_2/459
dspace.entity.typePublication
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