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dc.contributor.advisorBarbara Imperiali.en_US
dc.contributor.authorLuković, Elvedinen_US
dc.contributor.otherMassachusetts Institute of Technology. Dept. of Chemistry.en_US
dc.date.accessioned2010-05-25T20:41:53Z
dc.date.available2010-05-25T20:41:53Z
dc.date.copyright2009en_US
dc.date.issued2009en_US
dc.identifier.urihttp://hdl.handle.net/1721.1/55098
dc.descriptionThesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.en_US
dc.descriptionVita. Cataloged from PDF version of thesis.en_US
dc.descriptionIncludes bibliographical references.en_US
dc.description.abstractCatalyzed by kinases, serine/threonine and tyrosine phosphorylation is a vital mechanism of intracellular regulation and is involved in nearly every aspect of normal, as well as aberrant, cell function. With more than 500 protein kinases present in the human genome, the need for probes that can rapidly and selectively report the activity of a single kinase or a discreet subset of related kinases is crucial, particularly as researchers move to increasingly complex, and more relevant, systems to study the effects of dysregulated kinase behavior. We previously developed sulfonamido-oxine (Sox)-based fluorescent peptides following a P-turn focused (BTF) design. Upon phosphorylation of the Sox-containing peptide, the chromophore binds Mg + and undergoes chelation-enhanced fluorescence (CHEF). However, due to the BTF design limitation, only residues C- or N-terminal to the phosphorylated residue were used to specify the target kinase. To address this drawback, the recognition-domain focused (RDF) strategy, which also relies on CHEF, has been developed. In this approach, the Sox sensing moiety is introduced on the cysteine side chain (C-Sox), thereby allowing inclusion of extended kinase binding determinants, which are used to construct chemosensors for multiple Ser/Thr and Tyr kinases with greatly enhanced selectivity. Moreover, a high throughput mass spectrometry-based screening method that builds additional selectivity into RDF Sox-based probes for Ser/Thr kinases was also developed. Using this approach, it should be possible to construct short peptide probes with enhanced catalytic efficiency for virtually any kinase.en_US
dc.description.abstract(cont.) To expand the scope of CHEF-based sensors, beyond kinases that derive specificity from the short consensus sequence, a highly selective ERK sensor was prepared via semisynthesis by combining a recombinant kinase docking domain, PNT, with a synthetic sensing module that included the Sox chromophore. This probe was used to exclusively monitor ERK1/2 activity in unfractionated cell lysates in the absence of off-target kinase inhibitors. Furthermore, to improve the photophysical properties of the probes for cellular studies, we developed several oxine-based CHEF chromophores utilizing numerous approaches including the versatile click chemistry. The most promising derivative, p-bromophenyltriazoyl-oxine (Clk), displays a significant bathochromic shift in the excitation (15 nm) and emission (40 nm) maxima compared to Sox, and efficiently reports kinase activity when incorporated into peptides as a C-Clk residue. Together, the results presented in this thesis indicate the power that the CHEF-based sensors have to selectively, rapidly and with great sensitivity deliver new insight into the role of in vitro and endogenous kinases in various processes and under a variety of circumstances.en_US
dc.description.statementofresponsibilityby Elvedin Luković.en_US
dc.format.extent257 p.en_US
dc.language.isoengen_US
dc.publisherMassachusetts Institute of Technologyen_US
dc.rightsM.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.en_US
dc.rights.urihttp://dspace.mit.edu/handle/1721.1/7582en_US
dc.subjectChemistry.en_US
dc.titleDevelopment of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescenceen_US
dc.typeThesisen_US
dc.description.degreePh.D.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Dept. of Chemistry.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistry
dc.identifier.oclc588953263en_US


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