Thesis advisor: Jianmin Gao / My graduate research focuses on engineering and characterizing gramicidin A (gA), a natural fifteen-residue transmembrane channel peptide. It consists of D- and L- amino acids at alternate positions. gA is believed to fold into a β-helix in membranes, and two folded monomers at each leaflet of the lipid bilayer dimerize to form a transmembrane channel. gA shares the common features of other known membrane channels: a well defined structure that only allows the passage of specific ions, a gating mechanism, and a high abundance of aromatic residues. This dissertation includes two subprojects: I. Understanding Channel Formation: Aromatic Modifications of Gramicidin A Channel Ion channels are key elements in signaling and molecule transport, and therefore crucial for normal function of cells. Defective ion channels are known to be responsible for a number of diseases. Although hundreds of crystallographic structures of membrane proteins have been deposited into the PDB in the past few decades, our knowledge on this large family of proteins is still limited and mostly descriptive. Study of small peptides in model membranes is a good simplification of the more complex biological systems. In chapter 1, I will introduce my research using gA as a model system to understand the significant role of aromatic residues in membrane channel structure formation. Channel activities of these gA-Ar mutants were evaluated by ion leakage assays. The structure activity relationship of a library of gA mutants was discussed. The alternating chirality of amino acids was proven to be essential for gA channel activity. Several additional interesting observations are discussed. II. Towards Bacterium Specific Ion Channels: Solublized Gramicidin A as Potential Systemic Antibiotics The rapid development of multidrug resistance by pathogenic bacteria poses a serious threat to society and demands new antibiotics with different mechanisms. Often considered as a model transmembrane channel, gA also has proven antibiotic activities. The gA channel facilitates passive diffusion of water and monovalent cations (e.g. H+, Na+, K+) thus killing bacteria by disrupting the ion gradient across the cell membrane. However because of its poor solubility and high toxicity, its medicinal application as an antibiotic has been limited to topical reagents. A detailed understanding of gA allows rational optimization of the gA-WT to potential systemic antibiotics. Bacterial membranes are composed of a large fraction of anionic species, therefore, we hypothesize that strategic incorporation of cationic residues into gA will afford bacterium-specific toxicities. In addition, the charged residues will greatly improve the water solubility of gA. In chapter 2, I will introduce my research on developing soluble and bacterium specific gA as a potential systemic antibiotic. We firstly incorporated D-Lys at the C-terminus to obtain our first generation of gA based antibiotics. The best candidate (D-Leu10,12,14D-Lys gA) shows significantly increased water solubility (~ 1, 000 times) and therapeutic index (˃ 50 times). Modifications on the Lys side chain were then carried out to fine tune the antibiotic activities of these cationic gA. My research has pointed out a possible strategy to convert hydrophobic membrane channel peptides into potential systemic antibiotics. In addition to targeting the negative charges of bacterial membranes with cationic gA mutants, we proposed a novel strategy in which boronic acid is used to chase after the 1,2-diol substructure in the PG headgroup through boronate ester formation. Polyvalent display of boronic acids on a peptide scaffold results in enhanced binding with diols, showing promise of the boronate approach in the development of bacterium specific reagents. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_101463 |
| Date | January 2012 |
| Creators | Wang, Fang |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author, with all rights reserved, unless otherwise noted. |
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