Programmed mutagenesis and high-throughput methods to study protein recombination and epistasis

Abstract

Protein science, which includes studying extant proteins and designing novel proteins, requires a fundamental understanding of protein properties and principles. Decades of research have discovered properties of protein structure, dynamics, and function. In this work, we build upon this research to study protein recombination and epistasis with high- throughput methods. First, we develop two methods for high-throughput screening, a deep mutational library generation method (SPINE), and an automated neuron profiling technique. SPINE improves both comprehensive and uniform coverage for deep mutational library generation, and our automated neuron profiling technique measures neuromodulation, developmental effect, and baseline shifts, which we use to develop a sodium channel modulator. Secondly, we use these methods to study over 300,000 recombined proteins and 648,000 pairwise amino acid substitutions. We show that the interaction between the inserted peptide and recipient protein regulates recombination fitness. Additionally, we show that negative epistasis is wide-spread yet restricted to proximal residues, and positive epistasis is predominantly long-range interactions and enriched in evolutionarily conserved, function-defining, and clade-specifying residues.