(571l) The Beta Roll Peptide as a Reversible, Calcium Sensitive, and Modular Scaffold for the Engineering of Biomolecular Recognition

Beta roll peptide motifs are composed of repetitive calcium binding nonamers. These motifs are found in several proteases, lipases and haemolysins, and are defined by the consensus: GGXGXDXUX, where U is an aliphatic amino acid and X is any amino acid. Repeating peptide scaffolds have been increasingly utilized as a stable and modular interface for biomolecular recognition of target molecules. These repeat peptides can be concatenated to increase the area available to design biomolecular interaction, making possible the design of proteins that can bind targets of varying size. Calcium binding triggers the folding of the beta roll repeats such that the variable amino acids occupying the 7th and 9th position of every other nonamer form an interface on one side of the beta roll. Thus, the binding of calcium controls the assembly of the interface designed to bind its target, and in its absence, the target can be released from the beta roll. Here we present the characterization of the beta roll motif of a Serralysin as an independent functional scaffold on which one can design calcium controlled allosteric binding to small molecules, peptides and proteins. The functional folding of the beta roll peptide was determined using circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). The fluorescent resonance energy transfer (FRET) between the fluorophores of a cyan fluorescent protein (CFP)- beta roll – yellow fluorescent protein (YFP) fusion was used to monitor the calcium-induced folding and EGTA-induced unfolding of the beta roll. Further, our analysis explores the relevance of capping the beta roll to stabilize its hydrophobic core. The effect of various capping groups on the folding and reversibility of the beta roll are described. This reversible scaffold is potentially a useful platform on which to engineer allosterically controlled binding of target molecules for applications in biosensing, drug delivery, and stimulus-responsive hydrogels.