Abstract “Biomimetic Aminoacylation” Svetlana K. Tzvetkova Doctor of Philosophy, 2008 Graduate Department of Chemistry University of Toronto The accuracy of ribosomal protein synthesis depends on the fidelity of highly specific enzymes, aminoacyl tRNA synthetases, towards amino acid – tRNA pairs. These biological catalysts are responsible for activating the amino acids as aminoacyl adenylates and for their subsequent attachment to the 2’- or 3’-OH at the 3’-terminal of the correct tRNA to give aminoacyl-tRNA. Extended diversity in protein structure and function could be achieved if non-natural side chains can be introduced in protein synthesis. This requires that the acceptor stem of a tRNA molecule be synthetically aminoacylated. The most widely used methods for charging tRNA with non-natural amino acids involve multi-step synthesis of an aminoacyl-pCpA and its consequent enzymatic ligation to truncated tRNA. No direct route to these species has been reported. We have developed a method for direct biomimetic aminoacylation of the 3’-terminal hydroxyls of tRNA. Our approach shows to be promising in reactions leading to direct 2’- or 3’-O-aminoacylation of not only nucleosides and nucleotides but also RNA in general and tRNA in particular. The system we have developed provides: 1) efficient activation of the amino acids as aminoacyl phosphates, analogues of the enzymatic intermediates, and 2) specific recognition of the 3’-terminal of tRNA by lanthanide ions present in the reaction. The aminoacylating reagents used in our studies were carefully selected to provide handles to follow the reaction: UV absorbance, fluorescence spectroscopy and 19F NMR. Lanthanide (III) ions can play a role similar to a key part of the aminoacyl tRNA synthetases – they bring the aminoacyl close to the 3’-terminal of tRNA, in this case by forming a bis-bidentate complex with the aminoacyl phosphate and the 2’,3’-diol functionality of the 3’-terminal adenosine. This process relies on the specificity towards the unique 3’-terminal diol on tRNA, provided by the metal ion and the simultaneous complexation of the aminoacyl phosphate.

A METHOD BASED ON CONSERVED MULTIPLE AMINO ACID PROPERTIES TO PREDICT AMINO ACID SUBSTITUTIONS WHICH MAINTAIN THE PROTEIN STRUCTURE Avinash M. Baktula September 16, 2004 1-117 Directed by: Claire A. Rinehart, Doug McElroy and Sigrid Jacobshagen Department of Biology Western Kentucky University Proteins often contain several domains, each with a distinct structure. Such domains have evolved as units that, when combined in various arrangements, produce proteins of unique structure. This study was conducted to identify amino acid substitutions that don’t change structure. Amino acid properties which were conserved in proteins with identical structures were used to predict a set of amino acids profiles at each sequence position that can serve as viable substitutions. To test this analysis ten different protein sets were taken from the Conserved Domain Database of National Center for Biotechnology Information (NCBI). An amino acid index database of numerical indices representing various physicochemical and biochemical properties of amino acids were mapped onto the amino acid sequences in each dataset and these were used to select properties common to the proteins with the same structure. Based on these conserved properties, a substitution index percentage (SI%) was calculated to represent the relative ability of an amino acid to substitute at a given position and still maintain a protein structure. Amino acid profiles from different SI% ranges were used to create a set of substitutions into the consensus sequence of each dataset (AASCS). The AASCS from each SI% range were submitted to two validation programs, RPS-BLAST and PSI-PRED. The number of matches between the AASCS and the primary data set sequences for each SI% range was used to select the substitution profile ranges that best maintained the structure. It was concluded that amino acid, substitutions with SI% greater than 90% consistently conserved the structure of the protein. This method may prove useful in predicting the structure of proteins with induced mutations (site-directed mutagenesis), and in studies pertaining to protein engineering.

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Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaf 60). Also available via the World Wide Web.

Thesis (M.S.)--Florida State University, 2003. / Advisor: Dr. Emily Haymes, Florida State University, College of Human Sciences, Dept. of Nutrition, Food, and Exercise Science. Title and description from dissertation home page (viewed Feb. 26, 2004). Includes bibliographical references.

Thesis (M.S.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2010. Title from PDF t.p. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Kristi L. Kiick, Dept. of Materials Science & Engineering. Includes bibliographical references.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 226-231). Also available in electronic version. Access restricted to campus users.

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Hyperglycemia is associated with a higher risk for the development of cardiovascular diseases such as atherosclerosis and hypertension. Hyperglycemia-induced generation of reactive oxygen species and the endothelial dysfunction largely account for this phenomenon. Ergothioneine is a naturally occurring amino acid that is abundantly found in mushroom. Numerous benefits have been found associated with ergothioneine such as cation chelating, regulation of gene expression, improvement in immunity and bioenergetics, and of most concern its antioxidative property. The aim of this study was to investigate whether mushroom extract and synthetic ergothioneine can exert protective effect on endothelial cells against oxidative stress. Human umbilical vein endothelial cells served as the cell model. Pyrogallol, hydrogen peroxide and high glucose were used to create the oxidative stress condition in endothelial cells. Biochemical assay was used to measure the viability of the cells. It was found that only the mushroom extract could significantly reduce the cell death induced by pyrogallol. Both the mushroom extract and synthetic ergothioneine significantly decreased the cell death induced by high glucose. However, neither mushroom extract nor synthetic ergothioneine have any positive effect on hydrogen peroxide-induced cell death. These results indicated that mushroom extract and synthetic ergothioneine did exert certain level of protective effect on endothelial cells. However, this protective effect is relatively weak. Besides, it is still unclear if antioxidation is the sole mechanism accounting for the cytoprotective effect of ergothioneine. Further investigation is required to examine if other mechanisms are also involved. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences