Toward Life-Sustaining Structures: Creating a Library of Functional, Well-Ordered de novo Proteins

Abstract

Synthetic biology pushes the boundaries of what we know to be possible. Whereas traditional biology asks the question “what mechanisms has nature evolved?” in synthetic biology the question becomes “what is possible, but not yet tested by nature?” To probe outside the natural realm, one area of research is the creation of de novo protein libraries. Building off our knowledge of protein structure, we can design de novo libraries using a binary patterning strategy of polar and nonpolar amino acids. Our current research builds upon previous studies in the Hecht lab involving de novo libraries. In particular, we aim to combine two features found in previous libraries: the structural stability of the protein S-824 and the biological function of SynRescue proteins. To accomplish this, we create a new library of S-824 variants with specific mutations in the protein’s interior, designed to create potential catalytic sites while maintaining a highly ordered structure like the parent protein. After constructing this library, we test for biological function with auxotroph rescue experiments. Our results showed that proteins from this library are capable of rescuing E. coli cells with a conditionally essential gene knocked out, demonstrating that these native-like proteins, although completely synthetic and unevolved, can provide life-sustaining function.