High-Throughput Assessment of De Novo Protein Design: Generation of Molecular Probes for Guanine Exchange Factors

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Knowledge of the underlying principles of protein-protein interactions facilitates the design of proteins with novel functions that remain unobserved in nature and create new opportunities for scientists to dissect the molecular basis for diseases. Although significant advancements in the field have been made, evaluation of the computational design process by expressing proteins in vitro has been limited to small sample sizes due to constraints associated with gene synthesis. Recent advances in the field of oligonucleotide synthesis have enabled the high-throughput screening of designs with yeast display. We will harness this technology to assess new methods for packing amino acids at protein interfaces and to determine ideal score term targets for designed models. During this process, we will develop proteins that inhibit guanine exchange factors (GEFs), which are critical for cellular proliferation and have thereby garnered significant interest as therapeutic targets for cancer. We also aim to develop novel biosensors that are capable of reporting GEF activation by engineering a binding site into the SnapTag and HaloTag constructs. These results are expected to have an important positive impact for the field of protein engineering, ultimately providing new opportunities for the development of reagents that enable the scientific community to advance our understanding of biological processes.