Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2010
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| Online Access: | http://hdl.handle.net/1721.1/55098 |
| _version_ | 1811072687251914752 |
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| author | Luković, Elvedin |
| author2 | Barbara Imperiali. |
| author_facet | Barbara Imperiali. Luković, Elvedin |
| author_sort | Luković, Elvedin |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009. |
| first_indexed | 2024-09-23T09:10:15Z |
| format | Thesis |
| id | mit-1721.1/55098 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T09:10:15Z |
| publishDate | 2010 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/550982019-04-10T21:08:42Z Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence Luković, Elvedin Barbara Imperiali. Massachusetts Institute of Technology. Dept. of Chemistry. Massachusetts Institute of Technology. Dept. of Chemistry. Chemistry. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009. Vita. Cataloged from PDF version of thesis. Includes bibliographical references. Catalyzed 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. (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. by Elvedin Luković. Ph.D. 2010-05-25T20:41:53Z 2010-05-25T20:41:53Z 2009 2009 Thesis http://hdl.handle.net/1721.1/55098 588953263 eng M.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. http://dspace.mit.edu/handle/1721.1/7582 257 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemistry. Luković, Elvedin Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence |
| title | Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence |
| title_full | Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence |
| title_fullStr | Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence |
| title_full_unstemmed | Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence |
| title_short | Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence |
| title_sort | development of selective peptide and protein based reporters of kinase activity utilizing chelation enhanced fluorescence |
| topic | Chemistry. |
| url | http://hdl.handle.net/1721.1/55098 |
| work_keys_str_mv | AT lukovicelvedin developmentofselectivepeptideandproteinbasedreportersofkinaseactivityutilizingchelationenhancedfluorescence |