(164l) Designing Versatile Beta Roll Peptide Scaffolds

The Block V RTX “beta roll” peptide domain, located in the CyaA enzyme of B. pertussis, has been recognized in the past as a potential platform for ion-responsive protein capture, due to its ability to reversibly fold in millimolar concentrations of calcium ions. While the beta roll is a good candidate for selective binding, the applicability of the wildtype beta roll domain as a scaffold is narrow due to the limited number of designable positions on its binding face that can form protein-biomolecule interactions. We approached the challenge of designing novel lengthened and widened Block V domains by setting structural homology constraints and computationally predicting the thermodynamic conformations of residues. Utilizing this strategy, the most unfavorable residues in the new beta roll structures were identified and the surrounding areas were designed for optimal thermodynamic stability. Fluorescence resonance energy transfer (FRET) experiments have shown that computationally designed beta rolls with one and two additional N-terminal consensus loops fold in the presence of calcium ions. Concurrent results indicate that the conformation of residues in the additional N-terminal loops maintain an internal hydrophobic continuum within the structure. As has been shown for the recently characterized Block IV-Block V region of the RTX peptide, maintaining this region may help stabilize the scaffold and propagate the folding of additional loops. Widening the beta roll contributes similarly to an increase in thermodynamic stability. Computational models and crystallography data has indicated that greater thermodynamic stability of the structure in the absence of calcium is correlated to an increase in calcium binding affinity. The lengthening and widening processes can be conducted simultaneously to assemble structures that have the thermodynamic benefits corresponding to both structures. A larger binding face can partake in a greater diversity of protein-biomolecule interactions, which can be evolved to bind to the new scaffold via phage-assisted continuous evolution (PACE). We plan to express and crystallize novel lengthened and widened beta rolls to further validate these computational results, and to leverage this directed evolution pathway to improve the scaffold’s ability to bind biomolecular targets.