CARPE: Computationally Aided Rapid Protein Design

Directed evolution has become the forefront method of generating novel protein-peptide activity in protein engineering. However, current directed evolution protocols rely on random mutagenesis, consuming disproportionate amounts of time and resources. CARPE, computationally-aided rapid protein engineering, is a faster and more resource-efficient alternative to directed evolution. Using computationally proposed mutations to the active site of the Ste2 GPCR in Saccharomyces cerevisiae, we engineered Ste2 to recognize the kidney disease biomarker Cystatin-C instead of its native ligand. Evolutionary relationships were explored through pairwise alignments to identify binding residues with high Shannon entropy in Ste2. These target residues were computationally mutated to evolutionarily related amino acids to determine sets of mutations that optimized binding energy to Cystatin-C. To validate results, computationally-favorable mutations were induced in Saccharomyces cerevisiae upstream of mVenus to assess binding affinity between CARPE-mutated Ste2 and Cystatin-C.