(474c) Directed Evolution of Allosterically Regulated Beta Roll Subdomains for Biomolecular Recognition

Directed evolution has proven to be a highly effective approach for the engineering of biomolecular recognition into a broad range of protein scaffolds. There are several applications where allosteric control over the binding events would be beneficial, but the scaffolds used in these endeavors are generally incompatible with this design goal. We have identified the naturally existing beta roll subdomain as a potential scaffold onto which such functionality 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 reversible transition to a beta roll spiral structure that forms two parallel beta sheet faces, such that each beta strand in the face has two variable residues solvent exposed. We have extensively characterized a native beta roll subdomain along with various end-capping groups using a range of spectroscopic techniques in order to identify a minimal calcium-responsive beta roll unit. 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 randomized one face of this minimal beta roll unit and will present results of directed evolution experiments using bacterial surface display and ribosome display in order to identify beta rolls with biomolecular recognition capabilities. Directed evolution experiments are initially focusing on three model targets: streptavidin, troponin I (as a marker of cellular stress), and protective antigen (as a marker of biothreat pathogens). Potential applications of the beta roll in the context of biosensor development will also be discussed.