Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.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.by Jennifer Zhengzheng Yao.Ph.D
