Researcher(s)
- Phillip Moquin, Biochemistry, University of Delaware
Faculty Mentor(s)
- Zhihao Zhuang, Chemistry and Biochemistry, University of Delaware
Abstract
Ubiquitin (Ub) is a small (~8.6 kDa) 76 amino acid protein that is largely responsible for regulating various cellular processes including DNA damage repair, protein trafficking, and proteasomal degradation via ubiquitination, the process by which a Ub molecule is attached to a target substrate via an isopeptide linkage [1]. Monomeric Ub can further be ubiquitinated at its seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) as well as its N-terminus methionine, enabling the formation of polyUb chains. This property of Ub allows the construction of Ub activity-based probes, either branched or linear [2], which can be employed in proteomics experimentation to elucidate the mechanisms of Ub-mediated pathways. One such method of probe generation is α-halogen ketone-mediated chemical ubiquitination [1] which employs a chemical linker molecule to establish a linkage between substrates. Though α-bromo ketone linkers are widely used for this method of Ub probe generation [3], the usage of α-iodo ketone linkers has not been reported. Thus, this work examines the possibility of generating an iodine-substituted chemical linker to achieve improved ligation efficiency between Ub and any possible substrate, promoting greater yields when generating Ub probes. To produce the iodine-substituted linker molecule, a bromine-substituted non-hydrolyzable linker (NHL) was installed onto HA-tagged Ub1-75-MESNa, deprotected, and then incubated in an aqueous solution of potassium iodide. Preliminary proteomic mass spectrometry data collected from small-scale experimentation indicated successful halogen displacement via a Finkelstein reaction using this method. Ligation experiments were then conducted wherein deprotected HA-Ub1-75-NHL was treated with potassium iodide and a Cys-mutant Ub species to compare the ligation efficiency of the iodine-substituted linker to its bromine-substituted counterpart. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results suggest that the two linker species produce comparable ligations, though further testing is necessary to find conclusive answers.