De novo proteins from designed combinatorial libraries

Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins with interesting and important activities. Randomly generated sequences, however, rarely fold into well‐ordered proteinlike structures. To enhance the quality of a library, features of rational design must be used to focus sequence diversity into those regions of sequence space that are most likely to yield folded structures. This review describes how focused libraries can be constructed by designing the binary pattern of polar and nonpolar amino acids to favor proteins that contain abundant secondary structure, while simultaneously burying hydrophobic side chains and exposing hydrophilic side chains to solvent. The “binary code” for protein design was used to construct several libraries of de novo proteins, including both α‐helical and β‐sheet structures. The recently determined solution structure of a binary patterned four‐helix bundle is well ordered, thereby demonstrating that sequences that have neither been selected by evolution (in vivo or in vitro) nor designed by computer can form nativelike proteins. Examples are presented demonstrating how binary patterned libraries have successfully produced well‐ordered structures, cofactor binding, catalytic activity, self‐assembled monolayers, amyloid‐like nanofibrils, and protein‐based biomaterials.