Abstracts Category : Other

Investigating Chemical Modifications in a ComplexProteome

by Lisa Ann Crawford

Proteins are composed of the 20 naturally occurringamino acids and are further modified by a variety ofpost-translational modifications (PTMS). Naturally occurring aminoacids are diverse in structure and function. Catalytic amino acids,or nucleophilic amino acids, are of particular interest because oftheir contribution to chemical transformations in the cell.Synthetic covalent modification is a means to further functionalizeor diversify proteins. These modifications, or enhancements, allowfor improved understanding of protein structure, function andactivity. For instance, isotope labeling of amino acid side chainsin NMR studies enable investigators to study protein dynamics uponsubstrate or ligand binding. Fluorescence labeling is particularlyuseful to investigate protein cellular localization. Covalentmodification is a useful tool to investigate the relative level ofactivity for protein known to be regulated by PTMs. An importantfeature of covalent modification reactions is site specificity, asthis dictates the location, number of modifications, and proteintargets. Tyrosine is of particular interest because it is bothnucleophilic and aromatic. These characteristics contribute to theexistence of tyrosine residues in both the protein surface andhydrophobic cores. Tyrosine is incorporated into proteins at arelatively low frequency. Unlike lysine, which is ubiquitous onprotein surfaces, the low number of potential sites for generaltyrosine modifications makes it an attractive site for surfacebioconjugation modifications. A low number of surface modificationsis less likely to perturb native protein function. Bioconjugationreactions give access to functionalizing the surface of proteinswith moieties such as fluorophores, PEG, peptides, or drugs.Tyrosine is an attractive target for modifications because it isfound in the active sites of a variety of enzymes such assialidases, glutathione-S transferases, corticosteroid11-beta-dehydrogensase, DNA topoisomerase, and ferredoxin-NADP+reductase. Provided here is a survey of the known non-selective andselective synthetic chemical modification reactions for tyrosine.To investigate nucleophilic amino acids, Activity Based ProteinProfiling (ABPP) may be implemented to investigate the role ofthese residues. ABPP utilizes small molecule covalent probes as atool to selectively target enzymes in their active state. Toinvestigate a protein of interest (POI) (or class of proteins) byABPP, it is necessary to use a small molecule covalent probe thatselectively reacts with the POI over other proteins within theproteome. Due to this requirement, it is necessary to expand thecurrent ABPP probe toolbox to increase the coverage of whatproteins in the proteome may be studied. Inspired by findings inthe literature, our lab sought to explore the utility of variousaryl halides for implementation in ABPP probes to overcome thislimitation. This study revealed dichlorotriazine as a biologicallyrelevant and reactive electrophile. A focus was placed on aAdvisors/Committee Members: Eranthie Weerapana (Thesis advisor), Jianmin Gao (Thesis advisor).