Development of selective peptide- and protein-based reporters of kinase activity utilizing chelation-enhanced fluorescence

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2009

Author dc:creator

• Luković, Elvedin

Advisor dc:contributor.advisor

• Barbara Imperiali.

• Chemistry.

Statement dc:rights

• 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.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/55098

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/55098