htFuncLib: Designing Libraries of Active-site Multipoint Mutants for Protein Optimization.

Protein function relies on accurate and densely packed constellations of amino acids within the active site. The high density in the active site optimizes activity but reduces tolerance to mutations, thereby frustrating efforts to engineer or design new or dramatically improved activity. Introducing new activities may therefore require simultaneous multipoint mutations. Still, in a phenomenon known as epistasis, the outcome of combinations of mutations can differ significantly-and even reverse-the impact of the individual mutations, limiting predictability. To address these challenges we previously developed FuncLib, a method for the computational design of multipoint mutants in active sites. We recently extended FuncLib to enable the design of large combinatorial mutation libraries for high-throughput screening in a method called htFuncLib that generates compatible sets of mutations likely to yield functional multipoint mutants. htFuncLib enables scalable library design and experimental screening of hundreds and up to millions of active-site variants. This approach has generated thousands of active enzymes and fluorescent proteins with diverse functional properties. We have updated the FuncLib web server (https://FuncLib.weizmann.ac.il/) to support htFuncLib and introduced an electronic notebook (https://github.com/Fleishman-Lab/htFuncLib-web-server) for customizable library design, making those tools easily accessible for protein engineering and design. The new FuncLib web server enables reliable and scalable design of function for low-, medium- and high-throughput experiments through a single computational platform. We envision that this server will accelerate the optimization and discovery of function in enzymes, antibodies, and other proteins.