Thesis (Ph. D)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Aug 16

Thesis (Ph.D.) -- University of Texas at Arlington, 2008.

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 146-178).

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2009. / Includes bibliographical references (p. 57-66).

Ulm, Univ., Diss., 2007.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [123]-140).

Thiopeptides are a family of antimicrobial peptides that are characterized for having sulfur-containing heterocycles, and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The antimicrobial peptide micrococcin is a thiopeptide that is synthesized by the ribosome and undergoes several post-translational modifications (PTMs). Micrococcin is formed from a precursor peptide, TclE. TclE comprises an N-terminal leader (35-AA) that is crucial for recognition of the PTM machinery, alongside a C-terminal core sequence (14-AA) that undergoes multiple PTMs to acquire its antimicrobial activity. In the first series of modifications, the scaffold protein TclI binds the leader of TclE and presents the core of TclE to the modifying enzymes TclJ and TclN, facilitating the conversion of 6 cysteine residues into thiazoles. The work of this dissertation focuses on understanding the key interactions between the TclIJN protein complex and the precursor peptide TclE. By carrying out mutagenesis analysis on the leader peptide, I determined a minimal region of TclE that is required for thiazole installation. By doing bioinformatic analysis and copurification experiments, I determined that the TclI scaffold protein binds to the enzymes TclJ and TclN one at a time in dynamic equilibrium. I also further characterized the region of TclI that is important for coordinating these interactions and determined key residues that play a role for binding to its enzymatic partners. During my PhD, I had the opportunity to work on a few side projects that came up as I was working on plasmid construction for the Tcl project and working as a teaching assistant for the Microbial Genetics class (MMBIO360). During plasmid construction for protein expression of Tcl proteins, we recognized that there was room for improvement on transcriptional terminators, especially for the widely used T7 RNA polymerase. We engineered a set T7 terminators that are shorter and more efficient compared to previously reported T7 terminators, both in vivo and in vitro. As a teaching assistant for the MMBIO360 class, I had the opportunity to coordinate the work of undergraduate students in research-driven projects. We used the genetically tractable organism Agrobacterium fabrum to investigate flagellar motility. We carried out a near-saturating screen that led to the finding of four previously undescribed genes that are essential for motility in this organism. Another side project that also emerged from this class is investigating the genetics of streptomycin resistance in A. fabrum. Once paper from each of these three side projects are reprinted in Chapters 3, 4 and 5, respectively.

No description available.

Triazoles have been widely used as amide bond isosteres in chemical biology as linkers and to enhance proteolytic stability. The use of triazoles has grown exponentially since the discovery of the copper (I) catalysed alkyne azide cycloaddition reaction in 2002 as the reaction is solvent and functional group tolerant, and usually high yielding. The reaction is also orthogonal to reactions used in nature, meaning it has become a powerful coupling tool. In post-translational modification (PTM), proteins are modified by covalent attachment of functional groups to amino acid side chains. These PTM processes are generally thought to be dynamic and highly regulated by cell machinery, controlling protein function in response to stimuli. The ability to control function post protein synthesis allows organisms to have a smaller genome, which is advantageous as it reduces the energy required for DNA replication and repair. Research into the function of PTMs has been limited by the difficulty in generating recombinant proteins that bear a single PTM in a specific location. Although many elegant methods have been proposed that solve this problem, to date cysteine alkylation is one of the most successful techniques. For lysine PTMs, thia-lysine II (sLys) derivatives have been shown to be excellent mimics of lysine, where the only perturbation between the native lysine-containing analogue is the switch of a CH2 for S in the side chain. Biotin is a well-known PTM in biotin dependent carboxylases, where biotin is involved in CO2 transfer. Recently biotinylation has also been shown to be a PTM on many other proteins, however the role of biotinylation is not well understood. Biotin triazole III has been shown to be a good mimic of the biotin amide bond and retains excellent affinity to Avidin (Av). In Chapter 1 the effects of modification to the valeryl side chain, and orientation of the biotin triazole bond affect affinity to Av using ITC are investigated. Compounds III, V and VI are shown to have a KD < 120 pM, but further information on the binding affinity of these compounds could not be assessed by ITC. Biotin triazoles III-VI were also shown to be resistant to hydrolysis in serum, unlike the native biotin amide bond, which is hydrolysed by the enzyme biotinidase (BTD). Generation of amide sLys derivatives has been shown to be synthetically challenging. In Chapter 2, the synthesis and applications of chloromethyl-triazole biotin as a sulfhydryl selective alkylation reagent are investigated. The electron withdrawing nature of the triazole was proposed to give a ‘pseudo-benzylic’ halide α to the triazole, thus increasing reactivity. The controlled alkylation of peptides and proteins has shown that chloromethyl-triazole biotin shows enhanced reactivity over many commercial alkylation reagents and also gives good selectivity for cysteine. Alkylation of histone H4K12C gave the singly alkylated product, accompanied by low amounts of double alkylation. Biotinylation was confirmed by Western blot with anti-biotin. Due to the wide range of readily available functional azides, it was envisaged that halomethyltriazoles could be incorporated into other PTM mimics. In Chapter 3, efforts to expand the range of PTMs accessible using halomethyl-triazoles and further enhance the reactivity of chloromethyl triazoles by preparation of bromo- and iodomethyl triazoles are detailed. Synthesis of reagents to mimic malonylation, succinylation and GlcNAcylation PTMs is described and the reactivity of these halomethyl-triazole reagents is assessed. An alternate approach to the development of PTM mimics through cysteine propargylation and subsequent CuAAC coupling is also described in chapter 3. In conclusion, a series of new reagents have been developed to mimic protein PTMs through alkylation of cysteine. The reagents, which include biotin, GlcNAc, succinyl and malonyl mimics, are based on a halomethyl-triazole scaffold and have been successfully reacted with cysteine containing peptides and proteins.