Conjugation of proteins with ubiquitin (Ub) is a regulatory modification that touches all aspects of eukaryotic cell biology. Protein ubiquitination occurs via a common pathway that sequentially involves an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme, and an E3 Ub ligase. The UbcH5 family is the most highly conserved E2 family and is most often used for in vitro ubiquitination studies. A recent discovery made in the PI's lab that UbcH5 binds Ub non-covalently and that activated UbcH5~Ub self-assembles will be investigated using structural (NMR), biochemical, mutational, and cellular approaches. Integrity of the non-covalent UbcH5-Ub interaction has a profound effect on the ubiquitination products generated. Structures of the catalytically competent activated UbcH5~Ub and UbcH7~Ub (which neither forms self-assemblies nor poly-Ub chains) will be determined and compared. The functional ramifications of E2 self-assembly will be investigated with a variety of E3 ligases, using mass spectrometry to analyze products. Mutants that uncouple transfer of the first Ub to a target from subsequent steps of processive Ub chain formation will allow dissection of fundamental steps in ubiquitination.
The project will directly involve both graduate and undergraduate students who will be trained in diverse methods ranging from multi-dimensional NMR to yeast genetics. Each undergraduate student is paired with a graduate student, affording mentoring and training experience for the graduate students involved in this project. The undergraduate research experience is culminated by an oral presentation at a university-wide Undergraduate Research Symposium and a written thesis. The project will also form the foundation of a research seminar accessible to undergraduates given by the PI as outreach to undergraduate teaching institutions in the Pacific Northwest region to encourage students to participate in research activities. In most years, the P.I. offers students from regional institutions summer research experiences; projects relating to this project will be designed for such students. Research results will be disseminated in a timely fashion and will be published in peer-reviewed journals and deposited into the relevant public databases.
