Covalent attachment of ubiquitin, either as a monomer or as a polymer (polyubiquitin chains), serves as a signal that regulates the location, activity and/or degradation of many proteins. After serving as a signal polyubiquitin must be processed to monoubiquitin to maintain ubiquitin homeostasis. Isopeptidase T (IsoT) is a specialized deubiquitinating enzyme responsible for the regeneration of monoubiquitin from polyubiquitin chains. This role makes IsoT a central regulator of the ubiquitin system. The goal of this proposal is to understand how IsoT binds multiple isoforms of polyubiquitin chains by: 1) testing the polyubiquitin chain isoform binding selectivity of Isot; 2) determining how each of the four ubiquitin binding domains of IsoT contributes to the recognition of polyubiquitin chains; and 3) obtaining a molecular description of the recognition of polyubiquitin chains by IsoT using X-ray crystallography. Determining the substrate preference of IsoT and which domains are involved in binding each polyubiquitin chain isoform will provide insight into the general mechanism of polyubiquitin chain recognition and may aid in the development of pharmacological effectors of the ubiquitin system.