Intracellular communication via ubiquitin (Ub) signaling impacts all aspects of eukaryotic cell biology andregulates pathways critical to human development and viability. Aberrations or defects in Ub-signaling can result in numerous debilitating diseases including neurodegenerative diseases, infections, and cancer. Despiteremarkable progress over the decades, we still have only a rudimentary understanding of the molecular factorsand networks of interactions that govern the assembly of the proteins required for regulated Ub transfer andsignaling. The ability to intervene in diseases related to Ub-signaling requires a thorough understanding ofthese pathways at the molecular level. The basic scheme for Ub modification involves the concerted activitiesand interactions of several different proteins. This proposal focuses on expanding our understanding of acentral player in Ub-transfer reactions, the class of enzymes known as Ub-Conjugating Enzymes or E2s. Oncethought simply to shuttle Ub to the site of modification, emerging evidence suggests that E2s can play a pivotalrole in substrate recognition, determining the nature of Ub modification of a target protein, and even interactingwith and influencing the activities of proteins outside the traditional Ub-transfer pathway. There are ~40 E2s inthe human genome and most are thought to be directly involved in Ub transfer. Very little functionalinformation is available for the majority of these E2s. The Research Plan outlined in this proposal seeks tosubstantially expand our understanding of E2 function. Using various biochemical approaches, NMRspectroscopy, crystallography, and mass spectrometry, this project seeks to develop a molecularunderstanding of the factors that govern 1) the intrinsic activity of E2~Ub conjugates toward particularresidues, 2) E2~Ub recognition of substrates, and 3) and regulatory interactions of E2~Ub conjugates thatregulate proteins outside the main Ub-transfer pathway. We have adapted and developed new tools withwhich to identify proteins selectively modified by particular E2~Ub conjugates. We make use of a new "in coli"expression system that can reconstruct Ub-transfer pathways in E. coli. This system allows us to generateparticular E2~Ub conjugates and investigate their activity in cells without complications from competing Ub-transfer reactions. The new knowledge we seek to obtain will have a significant impact our understanding ofhuman E2 structure and function and greatly accelerate biological research involving ubiquitylation andubiquitin signaling.