(260e) Directed Evolution of the Intrinsically Disordered and Allosterically Regulated Beta Roll Subdomain for Biomolecular Recognition

Directed evolution is well-established as an effective approach for the engineering of de novo biomolecular recognition into a range of protein scaffolds. The majority of such scaffolds are highly ordered structures, such as antibody fragments. This is a beneficial property for biomolecules which are intended for therapeutic applications. In other contexts, however, it would be useful to use a scaffold with an allosterically-regulated structure which would enable triggered binding and release. We have identified the intrinsically disordered calcium-binding repeat-in-toxin (RTX) motif as a potential scaffold onto which recognition functionally can be engineered. The motif consists of tandem repeats of the sequence GGXGXDXUX, where U is an aliphatic amino acid and X is any amino acid. In the presence of calcium, the disordered peptide undergoes a transition to a beta roll structure consisting of two parallel beta sheet faces, such that each beta strand in the face has two variable residues that are solvent exposed. We believe that the two faces of the beta roll are suitable binding surfaces, and that calcium-induced structural formation can be used as an allosteric mechanism to control the beta roll structure and, thus, the formation of the engineered biomolecular recognition interface. The reversibility of the calcium binding suggests that the engineered biomolecular recognition will likewise be reversibly controllable. We have extensively characterized a native beta roll subdomain in order to identify a minimal calcium-responsive beta roll unit. We have randomized one of the beta sheet forming faces to create a library for use in directed evolution experiments using a circularly permuted outer membrane protein for bacterial cell surface display. In this session, we will discuss preliminary results of directed evolution experiments as well as potential applications of this system. Directed evolution experiments are initially focusing on three model targets: streptavidin, troponin I, and protective antigen. Streptavidin has been selected because it has been used previously as a target for directed evolution experiments so a variety of binding motifs are known as a basis for comparison. Troponin I and protective antigen have been selected as these are representative of the types of compounds that may be sought after in a future biosensor. Specifically, troponin I as a marker of cellular stress and protective antigen as a nontoxic marker of a biothreat pathogen.