Probing Folding Barriers in High Throughput using Proteolysis and Yeast Display

Abstract

A protein's primary sequence encodes its energy landscape, including the thermodynamic stability and dynamics of all accessible conformations. While the field has come a long way in understanding how thermodynamics are encoded, how the sequence encodes kinetic barriers is still poorly understood. Therefore, we are developing a novel combination of yeast display and proteolysis to select for high kinetic barriers. Proteolysis is an accessible and regularly used method that probes the energetics and dynamics of protein folding. Under the right conditions, proteolytic resistance arises solely from a large kinetic barrier to unfolding, allowing us to measure barrier heights directly. We have coupled proteolysis with yeast display, creating a high-throughput approach to select and monitor unfolding barrier heights, allowing us to systematically investigate how kinetic barrier height varies with changes in protein sequence. In addition to expanding our basic knowledge of protein folding, exploration of the relationship between sequence and kinetic barriers will allow for the development of long-lived drugs and shelf-stable detergents or a greater understanding of how pathogenic protein states arise.