Characterization of Functionally Active Proteins from a Combinatorial Library of Novel Sequences

Abstract

The field of protein design aims to understand the relationship between the structure of large molecules and their function in order to gain control over them. De novo protein design is a popular approach for tapping into the vast number of sequences not already employed by nature. The Hecht Lab has aspired to probe this unused sequence space on a large scale by generating a combinatorial library of de novo proteins anticipated to fold into four-helix bundles. This study presents the characterization of four sequences from the Next Generation Library—a library consisting of right- and left-handed subsets of four-helix bundles—that had been found to sustain growth of Δfes auxotrophic cells on minimal media. These sequences were found to be monomeric by size-exclusion chromatography and alpha-helical by circular dichroism. Thermal denaturation experiments of a previously unreported construct, L4HB08, demonstrated the impressive stability seen in other library sequences by remaining folded beyond 94 ˚C. Two of these sequences were chosen to investigate whether the Δfes auxotroph rescue is performed by the sequences’ protein or RNA—a distinction made using stop and frameshift mutations. The lack of a well-behaved negative control resulted in inconclusive results, so further analysis of the library is necessary to evaluate the relationship between enhanced stability and functionality in Δfes and other auxotrophs.