Chemical and enzymatic tools to study proteins in their native cellular environment

A detailed understating of living systems requires methods to probe molecular processes in cells and whole organisms. A set of technologies that combines chemical and genetic probes have been developed to address the need for dynamic and noninvasive assay of biological processes. In addition to be able to visualize the localization, trafficking, and turnover of individual proteins, strategies that allow the tagging, and imaging, and identification of entire proteomes have also offered valuable insights into disease biology. Since protein visualization serves as a complement to protein identification, this thesis first describes the development of a protein labeling technique that is able to specifically target diverse fluorophores to proteins inside live cells. The methodology uses the E. coli lipoic acid ligase (LplA) that we have engineered to accept and ligate an azide functional handle onto a 13-amino acid ḺplA a̲cceptor peptide (LAP). Subsequent derivatization of the azide with fluorophores functionalized with cyclooctyne via strain-promoted azide-alkyne cycloaddition allowed us to target many bright and photostable fluorophores that could be used in super resolution imaging. Due to the numerous applications to which cyclooctynes are being applied, our observation of the behavior of different cyclooctynes inside cells should also prove useful to the protein labeling community and beyond. For protein identification, we describe our work of engineering and using LplA to site-specifically target a benzophenone photocrosslinker. Our observations led us to the conclusion that although benzophenone is generally regarded as the more efficient and specific photocrosslinker than aryl azide and diazirine, its high geometric constraint to its proximal crosslinkable C-H bonds may decrease its crosslinking yield. Knowing the protein structure and amino acid environment surrounding benzophenone could help in choosing the most optimal position for the photocrosslinker. Finally, in a different crosslinking approach, we discuss our effort towards using a promiscuous peroxidase enzyme that generates biotin-phenol radicals to study membrane protein topology.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2013

Author dc:creator

• Yao, Jennifer Zhengzheng

Advisor dc:contributor.advisor

• Alice Y. Ting.

• 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/1721.1/82328

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/82328