(169a) Structure/Function Analysis for the Optimization of the Beta Roll Motif as a Novel Scaffold for Engineering Biomolecular Recognition

A broad range of successfully utilized protein scaffolds exist for the purpose of creating novel biomolecular recognition molecules using combinatorial methods in protein engineering. These scaffolds, however, typically bind in an almost irreversible fashion, thus limiting some of their potential applications. In contexts such as biosensing or smart chromatography systems, it is often essential that a scaffold be capable of binding and subsequently releasing its target while keeping the scaffold intact. We have previously discussed how the beta roll forming repeats-in-toxin (RTX) motif can act as a novel scaffold capable of allosterically-regulated molecular recognition. 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 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 identified the following set of structural and functional properties which are essential to investigate in order to optimize the use of the beta roll as biomolecular recognition scaffold: (1) End-capping requirements for calcium responsive folding, (2) Effect of number of repeats on calcium affinity and folding reversibility, (3) Solid surface immobilization as an alternative to protein-based caps. In ordered to study the end-capping requirements we have used a range of spectroscopic techniques to study the effect of several natural and unnatural caps on folding behavior. To study the effect of repeat number, we have implemented a recursive ligation technique to create constructs with various repeat lengths. Finally, to study surface immobilization we have created beta roll constructs with cysteine-modified termini and have used a quartz crystal microbalance to study their calcium responsiveness. Results of these structure-function studies as well as preliminary directed evolution experiments using the optimal scaffold will be presented.