Engineering the Beta Roll Peptide to Participate in Useful Biomolecular Interactions
Series: CBE Departmental Seminars
Location: Elgin Room (E-Quad A224)
Date/Time: Wednesday, December 12, 2012, 4:00 p.m. - 5:00 p.m.
For the last several years we have been exploring the beta roll forming repeats-in-toxin (RTX) peptide as a unique scaffold for protein engineering studies as it has the useful feature of being instrinsically disordered in the absence of calcium, and it folds into a well-defined 3-D structure in the presence of calcium. We have extensively characterized this conformational change using a variety of techniques. We have explored the capping requirements for the scaffold, we have made synthetic peptides with a repeated consensus sequence, and we have concatenated beta rolls together to explore how this impacts the folding of the peptide. We have also immobilized the peptides to explore their functionality when tethered. Now we have begun to engineer the beta roll peptide for useful functional applications. In the first line of work, we have engineered one face of the beta roll with leucine side chains. This enables the beta roll to dimerize in the presence of calcium and we have shown that this can serve as a stimulus-responsive cross-linking domain for use in protein hydrogel formation. We have also shown that a consensus sequence of beta roll domains reversibly precipitate in response to calcium and we have explored this as a novel protein purification tag that is more useful than the commonly used elastin-like peptide sequences. Finally, we have been working to engineer beta roll mutants with affinity for different target proteins using a variety of selection techniques including cell-surfaces display, phage display and ribosome display. The most recent results of these efforts will also be presented.