Thesis advisor: Eranthie Weerapana / This thesis includes two projects: “Bacteria-selective borono-peptides” and “A split ligand for lanthanide binding: facile evaluation of dimerizing proteins”. In both projects, de novo designed molecules were synthesized, optimized and incorporated into peptides. These synthetic molecular tools allow selective targeting of bacterial cell membranes and analyzing the dynamic associations of membrane-embedded proteins. 1. Bacteria-selective borono-peptides As the antibiotic resistance continues to grow, bacterial infection becomes one of the major threats to global public health. Currently, almost all the bacteria targeting strategies employ non-covalent driving forces, including charge-charge interactions, hydrophobic interactions and the formation of hydrogen bonds, to achieve bacterial selectivity. Towards novel bacteria targeting molecules, we have recruited reversible covalent chemistry in the development of bacteria-selective peptides. Targeting the diol-rich environment of a bacterial surface, we have designed and synthesized several unnatural amino acids that contain boronic acid moieties. Taking advantage of the boronic acid-diol reaction and multivalency effect, our borono-peptides are found to selectively recognize bacteria over mammalian cells. The sensitivity of the binding event to carbohydrate competitors gives a safe and facile approach to regulate molecular association with bacterial cells. This design may find applications in the fields of bacterial detection, imaging and antimicrobial drug delivery. 2. A split ligand for lanthanide binding: facile evaluation of dimerizing proteins Protein dimerization is a ubiquitous phenomenon in biology and plays a critical role in transcription regulations and various signaling processes. Methods that allow facile detection and quantification of protein dimers are highly desirable for evaluating protein dimerization in physiology and disease. Meanwhile, luminescence of lanthanides is attractive for biological applications due to its long lifetime and sharp emission profiles. We have developed a split lanthanide binding ligand that allows facile evaluation of dimerizing proteins. The fast lanthanide–ligand (dis)association allows us to monitor the dynamic behavior of dimerizing proteins. We have demonstrated the successful application of our assay on both soluble and transmembrane proteins in complex biological milieu. The split lanthanide ligand is cysteine reactive, and therefore should be readily applicable to a variety of proteins of interest. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Primary cilia are sensory organelles on many postmitotic cells. The ciliary membrane is continuous with the plasma membrane but differs in its phospholipid composition with phosphatidylinositol 4,5-bisposphate (PIP₂) being much reduced toward the ciliary tip. In order to determine the functional significance of this difference, we used chemically induced protein dimerization to rapidly synthesize or degrade PIP₂ selectively in the ciliary membrane. We observed ciliary fission when PIP₂ was synthesized and a growing ciliary length when PIP₂ was degraded. Ciliary fission required local actin polymerisation in the cilium, the Rho kinase Rac, aurora kinase A (AurkA) and histone deacetylase 6 (HDAC6). This pathway was previously described for ciliary disassembly before cell cycle re-entry. Activating ciliary receptors in the presence of dominant negative dynamin also increased ciliary PIP₂, and the associated vesicle budding required ciliary PIP₂. Finally, ciliary shortening resulting from constitutively increased ciliary PIP₂ was mediated by the same actin – AurkA – HDAC6 pathway. Taken together, changes in ciliary PIP₂ are a unifying point for ciliary membrane stability and turnover. Different stimuli increase ciliary PIP₂ to secrete vesicles and reduce ciliary length by a common pathway. The paucity of PIP₂ in the distal cilium therefore ensures ciliary stability.