Engineering the Beta Roll Peptide for Biotechnology Applications

Engineering the Beta Roll Peptide for Biotechnology Applications

Beyza Bulutoglu¹, Kevin Dooley² and Scott Banta¹

¹ Chemical Engineering Department, Columbia University, New York, NY

² Center for Engineering in Medicine, Harvard Medical School, Boston, MA

In nature, there are many proteins and peptides that are natively or intrinsically disordered. Often these peptides gain structure upon interaction with other proteins or molecules. Using protein-engineering tools, we have explored one of such peptide: the β-roll. The β-roll is a unique intrinsically disordered, allosterically–regulated peptide motif. This peptide is isolated from the repeats-in-toxin (RTX) domain and is intrinsically disordered in the absence of calcium. In calcium rich environments, the peptide binds Ca⁺⁺ ions and folds into a β-roll secondary structure composed of two parallel β-sheet faces. We have extensively characterized this calcium responsive RTX domain and evaluated its potential as a cross-linker for hydrogel formation and as an alternative scaffold for biomolecular recognition.

By rationally designing the two faces of the folded beta helix to contain leucine residues, we have created an environmentally–responsive cross-linking domain, genetically fused to an α-helical leucine zipper, capable of self-assembly only in the presence of calcium. We characterized this "double-face" leucine-rich RTX domain and evaluated its mechanical and biophysical properties using various techniques such as circular dichroism and michrorheology. To further investigate its cross-linking capability, we constructed concatemers of this β-roll with maltose binding protein (MBP) and demonstrated that the engineered β-roll peptide can mediate calcium-dependent proteinaceous hydrogel formation without the need for other cross-linking moieties.

In addition, we proposed that the β-roll peptide would be a suitable binding scaffold combining molecular recognition with a simple elution mechanism. This will be of great interest in the bio-separation area as well as other areas of biotechnology, where binding events can be controlled by benign environmental perturbations. Engineering the β-roll results in an allosterically–regulated scaffold, where the folding function can be decoupled from the binding function. Thus, this peptide can operate as a protein switch for biomolecular recognition, which can be mediated by simply changing the calcium concentration, allowing control over the binding behavior of the molecules.

For these purposes, we constructed a library containing mutated residues in eight positions, which form the beta sheet face upon folding. This way, one face of the folded β-roll is fully randomized. Using the ribosome display technique, we selected new β-roll peptides showing affinities for various targets including lysozyme and the Fc region of an IgG antibody. Isothermal titration calorimetry (ITC) was utilized to quantitatively determine the thermodynamic parameters of interactions in solution between the mutants and their targets. The latest results on the molecular recognition capabilities of the candidates obtained via this method will be presented.