Probing unfolding barriers in high throughput.

Abstract

Energy barriers between protein states determine the rate at which a folded protein makes excursions to and from any high energy states. These may include functional states or potentially harmful states. Although barriers between states are important features of a protein's energy landscape, we do not understand how they are encoded in a protein's primary sequence. Therefore, we have developed an assay coupling quantitative proteolysis with a high throughput display system to probe the effect of sequence changes on a protein's unfolding barrier. Under certain conditions, proteolytic resistance arises solely from a large kinetic barrier to unfolding, allowing us to measure barrier heights directly. In addition to expanding our basic knowledge of protein folding, exploration of the relationship between sequence and kinetic barrier heights may contribute to a greater understanding of how pathogenic protein states arise and may aid to the development of long-lived, shelf-stable protein drugs.