PROJECT SUMMARY/ABSTRACT Natural evolution has produced a stunningly diverse array of proteins that perform an equally diverse set of molecular functions in living organisms. These proteins—which constitute the primary raw material from which we might seek to develop new protein reagents—have been honed over the course of their mutational history to meet specific functional challenges. As a result, turning them to new functions by rational means often proves problematic: their expression and/or stability are compromised by our reengineering attempts, and their relic functionality is at odds with our intended use. De novo protein design, which uses sophisticated computer algorithms to identify stable sequence:structure pairings without relying on native templates, can create protein folds never before seen in Nature, and thus offers an alternative source of protein scaffolds for functionalization. We recently reported the development of new algorithms for de novo design of a particular class of proteins— circular tandem repeat proteins or cTRPs—whose modular, self-reinforcing symmetrical architecture offers advantages that include high stability, tunable geometry, and switchable oligomeric state. We hypothesize that de novo designed proteins in general, and these designed cTRPs in particular, will prove to be a valuable source of protein scaffolds for downstream application. Our aims in this proposal are first, to further develop our algorithms in order to design and experimentally validate a diverse set of cTRP scaffolds of varied size and topology; and second, in collaboration with clinical colleagues here at the Hutchinson Center, to evaluate these designs as scaffolds for presentation of functional domains with precisely controlled symmetry and geometry. Our collaborators will test these designed constructs in cellular assays with the goal of speeding the development of cellular therapies. Successful completion of this research will lead to (1) improved protein design algorithms that have been rigorously validated across a range of topologies and are available to the research community; (2) a family of stable and robust protein scaffolds for downstream functionalization, all of whose members have been structurally and biophysically characterized; (3) a set of useful protein reagents for biomedical applications.