Overline: BIOCHEMISTRYEditor's Summary: Artificial proteins target SUMO SUMOylation is the covalent attachment of SUMO 1, SUMO 2, SUMO 3, or combinations thereof to target proteins. This posttranslational modification controls protein function and localization. Hughes et al. screened a library of artificial proteins called Affimers to identify those that bound to SUMO. By incorporating a negative selection step to remove Affimers that bound to SUMO 1, the authors identified Affimers that recognized SUMO 1, SUMO 2/ 3 (SUMO 2 and SUMO 3, which are almost identical), or all three isoforms. Biochemical and cellular assays showed that these SUMO specific Affimers (S Affs) did not interfere with SUMO conjugation or deconjugation but did inhibit a cellular stress response that required SUMO mediated protein interactions. In addition to generating S Affs that will be useful tools for studying SUMO dependent cellular processes, this study also shows the applicability of this technology for generating reagents that interfere with specific protein protein interactions, which are useful for basic research and potentially for clinical development. Furthermore, through structural analysis and molecular modelling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO1 mediated interactions or interactions mediated by either SUMO2 or SUMO3. Not only will these reagents enable investigation of the biological roles of SUMOylation, the Affimer technology used to generate these synthetic binding proteins could be exploited to design or validate reagents or therapeutics that target other protein protein interactions.