(160av) Computational Design for the Lengthening and Widening of Beta Roll-Forming Peptides for Emerging Biotechnology Applications

Environmentally responsive peptides are important building blocks in synthetic biology for use in emerging biotechnology applications. The intrinsically disordered isolated Block V repeats-in-toxin (RTX) peptide domain of adenylate cyclase (CyaA) from Bordetella pertussis reversibly folds into a beta-roll structure in the presence of calcium ions¹. This conformationally dynamic peptide has been previously used as a scaffold for stimulus-responsive biomolecular recognition and self-assembling hydrogel formation^1-3, but more structural characterization is needed to increase affinity for greater protein capture. We used computational design methods in Rosetta⁴ to iteratively increase the width and length of the beta-roll binding face (PDB ID: 5cvw)⁵. By building loops using homologous sequences of the beta roll, we optimized the thermodynamic stability of conformations, accounting for interactions between the peptide and calcium ions. With a combination of this thermodynamic analysis and homology modeling, the most recent data indicates that the beta-roll functions most similarly to the wildtype with the top six beta strands lengthened by two amino acids, increasing the total number of binding residues in each strand from three to five. Additionally, a single reiteration of the third and fourth consensus sequences from the N-terminus in the wildtype proved to be the most energetically favorable lengthened scaffold. Currently, we are expressing the designed wider and longer beta-roll peptides in E. coli and characterizing those mutants using a FRET system⁶ with plans to create a beta-roll that is both wider and taller than the wildtype. These structural modifications that increase the size of the binding interface introduce the possibility of encoding additional residues onto a beta-roll interface, creating a larger interface for the engineering of biomolecular recognition.

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