Caged phosphopeptides and phosphoproteins : probes to dissect the role of phosphorylation in complex signaling pathways

Protein phosphorylation is a central regulatory mechanism in signal transduction pathways and cellular migration. Current genetic strategies for the study of phosphorylation, including gene knockout and point mutation, are limited in providing temporal information. As a complement to these techniques, the synthesis and semisynthesis of probes that enable researchers to observe the downstream effects of kinase-mediated phosphorylation in "real time" are presented in this thesis. The release of a physiologically-relevant concentration of a phosphopeptide with temporal and spatial control is accomplished by the photolysis of a photolabile precursor, a caged phosphopeptide. The synthesis and application of NI-Fmoc-protected 1-(2-nitrophenyl) ethyl (NPE) caged phosphothreonine, serine, and tyrosine building blocks facilitate the straightforward assembly of any caged phosphopeptide through Fmoc-based solid phase peptide synthesis. Removal of the NPE caging group by irradiation with long-wavelength UV light generates a concentration burst of the corresponding phosphopeptide. In addition, the installation of a caged phosphoamino acid into a full-length, multi-domain protein, the cellular migration protein paxillin, is described. A strategy, which is applicable to any expressible protein target, is detailed for the semisynthesis of a paxillin variant with a caged phosphorylated tyrosine at residue 31 of the 557-residue protein using native chemical ligation.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Vogel, Elizabeth Maura

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/1721.1/38621

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/38621